本文關(guān)鍵詞： 肽脫甲酰基酶 PDF64 細(xì)胞增殖 細(xì)胞凋亡 自噬 Akt/mTOR通路　出處：《華東師范大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

【摘要】：背景:在過去幾十年中,細(xì)菌肽脫甲酰酶(PDF)在致病性微生物中被認(rèn)為是抗菌藥物理想的潛在靶標(biāo),并且有很多的細(xì)菌PDF抑制劑被廣泛報(bào)道。然而,最近研究發(fā)現(xiàn)肽脫甲酰酶也存在于真核生物中,HsPDF(人線粒體PDF)由細(xì)胞核合成而在線粒體中發(fā)揮功能,同樣催化新生多肽的去甲�；�。HsPDF在乳腺癌、結(jié)腸癌和肺癌等多種癌組織中高表達(dá),其抑制劑表現(xiàn)出良好的體內(nèi)外抗腫瘤活性,HsPDF有望成為極具潛力的新型抗腫瘤靶標(biāo)。本課題組以目前處于臨床試驗(yàn)的細(xì)菌PDF靶標(biāo)化合物L(fēng)BM415結(jié)構(gòu)為基礎(chǔ),設(shè)計(jì)合成了一系列結(jié)構(gòu)類似的四氫吡咯類衍生物,初步的研究發(fā)現(xiàn)這些化合物存在不同程度的細(xì)菌PDF體外抑制活性(IC50=2～12 nM)及腫瘤細(xì)胞增殖抑制活性。目的:本論文主要通過體內(nèi)外實(shí)驗(yàn)對(duì)這一系列化合物的抗腫瘤活性進(jìn)行評(píng)價(jià),并對(duì)其作用機(jī)制進(jìn)行初步研究。體外實(shí)驗(yàn):首先我們通過CCK-8法對(duì)本課題組合成的一系列LBM415結(jié)構(gòu)類似物進(jìn)行抗腫瘤活性篩選及IC50值的測(cè)定,最終得到活性較好的化合物PDF64。接著我們運(yùn)用CCK-8法測(cè)定PDF64對(duì)不同組織來源腫瘤細(xì)胞的抑制活性,實(shí)驗(yàn)發(fā)現(xiàn)PDF64具有廣譜的抑制腫瘤細(xì)胞生長(zhǎng)的作用(IC50=2～11μM),并呈現(xiàn)一定的腫瘤細(xì)胞選擇性。體內(nèi)實(shí)驗(yàn):我們利用HCT116細(xì)胞裸鼠荷瘤模型測(cè)定了 PDF64的體內(nèi)抗腫瘤活性,結(jié)果顯示,高劑量PDF64在體內(nèi)表現(xiàn)出優(yōu)異的抗腫瘤作用,但也存在較大的毒性。150 mg/kg PDF組的平均抑瘤率為85.83%,優(yōu)于等劑量的陽性化合物actinonin(平均抑瘤率73.53%),但其毒性效應(yīng)導(dǎo)致荷瘤裸鼠的死亡,最后實(shí)驗(yàn)組僅有兩只荷瘤裸鼠存活。各組腫瘤組織細(xì)胞增殖活力指標(biāo)Ki67的免疫組化檢測(cè)發(fā)現(xiàn),PDF64組Ki67陽性細(xì)胞數(shù)目明顯低于對(duì)照組,甚至低于Actonion組。各組裸鼠心臟、肝臟、腎臟、肺、和脾臟的HE染色分析沒有發(fā)現(xiàn)PDF64對(duì)相關(guān)臟器的明顯損傷效應(yīng),因此高劑量PDF64的毒性效應(yīng)還需要我們做進(jìn)一步的探討。隨后我們又進(jìn)行了低劑量PDF64的抗腫瘤活性研究,50mg/kgPDF64組的平均抑瘤率為83.02%,優(yōu)于等劑量actinonin組(平均抑瘤率為63.47%),整個(gè)實(shí)驗(yàn)周期均未出現(xiàn)小鼠的死亡,體重也沒有較明顯的變化,實(shí)驗(yàn)結(jié)果提示荷瘤裸鼠能耐受50 mg/kg的PDF64給藥劑量,且低劑量的PDF64同樣具有非常優(yōu)異的抗腫瘤活性。機(jī)制研究:為了探討PDF64的抗腫瘤作用機(jī)制,我們首先分析了該化合物對(duì)細(xì)胞凋亡的影響。Annexin V-FITC凋亡實(shí)驗(yàn)發(fā)現(xiàn)PDF64對(duì)Jurkat和HCT116細(xì)胞有不同程度的凋亡誘導(dǎo)效應(yīng),PDF64對(duì)Jurkat細(xì)胞的凋亡誘導(dǎo)效應(yīng)明顯高于HCT116,并具有時(shí)間劑量依賴性。進(jìn)一步分析發(fā)現(xiàn),PDF64能明顯降低Jurkat細(xì)胞線粒體膜電位,而對(duì)HCT116細(xì)胞的線粒體膜電位影響較小,類似地PDF64能誘導(dǎo)ATP生成量下降,但對(duì)Jurkat細(xì)胞的作用更明顯。在凋亡相關(guān)標(biāo)志蛋白表達(dá)的研究中發(fā)現(xiàn),Jurkat細(xì)胞經(jīng)PDF64作用72 h后,其抗凋亡蛋白Bcl-2的表達(dá)量明顯降低,Caspase-3明顯被激活,并檢測(cè)出PARP-1剪切形式。而在HCT116細(xì)胞中,抗凋亡蛋白Bcl-2的表達(dá)量并沒有明顯變化,也沒有檢測(cè)出Caspase3的激活形式,但檢測(cè)出PARP-1剪切形式。系列實(shí)驗(yàn)表明PDF64可能介導(dǎo)了Jurkat細(xì)胞的線粒體凋亡途徑,而PDF64對(duì)HCT116細(xì)胞的抑制效應(yīng)則可能與其他的途徑有關(guān)。我們采用CFDA SE試劑盒檢測(cè)PDF64對(duì)腫瘤細(xì)胞分裂增殖的影響,實(shí)驗(yàn)發(fā)現(xiàn)PDF64均能時(shí)間、劑量依賴性的抑制HCT116、Jurkat細(xì)胞的分裂增殖。細(xì)胞周期實(shí)驗(yàn)發(fā)現(xiàn),PDF64對(duì)Jurkat和HCT16細(xì)胞的細(xì)胞周期分布均沒有明顯影響,在周期相關(guān)標(biāo)志蛋白表達(dá)的研究中發(fā)現(xiàn),PDF64并沒有使PCNA、CyclinD1和Cyclin D3的表達(dá)量產(chǎn)生明顯變化。PDF64能有效地誘導(dǎo)Jurkat細(xì)胞進(jìn)入線粒體凋亡途徑,而對(duì)HCT116細(xì)胞的凋亡效應(yīng)則不明顯,我們推測(cè)PDF64對(duì)HCT116細(xì)胞作用可能是通過自噬來實(shí)現(xiàn)的。通過MDC染色結(jié)合流式細(xì)胞術(shù)實(shí)驗(yàn),我們發(fā)現(xiàn)PDF64均能有效地誘導(dǎo)HCT116、Jurkat細(xì)胞的自噬效應(yīng),且對(duì)HCT116細(xì)胞的自噬誘導(dǎo)效應(yīng)更為明顯。自噬標(biāo)志分子LC3 Western blotting檢測(cè)發(fā)現(xiàn),經(jīng)PDF64作用72h后,在HCT116及Jurkat細(xì)胞中LC3-Ⅱ表達(dá)量明顯增加。已有的實(shí)驗(yàn)結(jié)果說明,PDF64誘導(dǎo)了HCT116和Jurkat細(xì)胞的自噬效應(yīng)。MAPKs及AKT/mTOR在腫瘤的發(fā)生發(fā)展中起重要作用,為了進(jìn)一步分析PDF64的抗腫瘤作用機(jī)制,我們對(duì)相關(guān)通路的蛋白及其激活形式的水平進(jìn)行了Western blotting檢測(cè)。結(jié)果顯示,在HCT116細(xì)胞中,PDF64能明顯抑制p38、mTOR、Akt等蛋白的激活,而在Jurkat細(xì)胞中,PDF64也顯著抑制了AKT/mTOR信號(hào)通路中mTOR的磷酸化水平,而對(duì)其他蛋白的磷酸化及非磷酸化形式影響較小。另外PDF64誘導(dǎo)了轉(zhuǎn)錄因子c-Myc蛋白表達(dá)量呈劑量依賴性的下降。已有的數(shù)據(jù)表明AKT/mTOR,p38 MAPK信號(hào)通路及轉(zhuǎn)錄因子c-Myc可能參與了PDF64引起腫瘤細(xì)胞的增殖抑制、凋亡和自噬效應(yīng),但其具體機(jī)制因細(xì)胞類型的不同又略有差異。實(shí)驗(yàn)結(jié)論:總之,四氫吡咯類化合物PDF64具有良好的體內(nèi)外抗腫瘤活性,對(duì)Jurkat腫瘤細(xì)胞具有促凋亡、細(xì)胞增殖抑制及自噬效應(yīng),對(duì)HCT116細(xì)胞則主要誘導(dǎo)自噬效應(yīng)。其抗腫瘤作用可能與AKT/mTOR通路及c-MYC蛋白相關(guān),但具體機(jī)制及在兩種細(xì)胞中效應(yīng)的異同尚需進(jìn)一步研究。本研究為此類藥物的設(shè)計(jì)合成提供了實(shí)驗(yàn)依據(jù),為PDF抑制劑的抗腫瘤活性研發(fā)提供了理論基礎(chǔ)。
[Abstract]:Background: in the past few decades, the bacterial peptide deformylase (PDF) in pathogenic microorganisms is considered as a potential target for antibacterial drug ideal, and there are many bacterial PDF inhibitors have been widely reported. However, recent studies found that peptide deformylase also exist in eukaryotes, HsPDF (human mitochondrial PDF) by nuclear synthesis function in mitochondria, the same catalytic hydroformylation of nascent polypeptide to.HsPDF in breast cancer, high expression of a variety of carcinoma of colon and lung, the inhibitor showed good anti-tumor activity in vitro and in vivo, HsPDF is expected to become a new anticancer target potential. This topic in group is currently in the bacterial PDF compounds with LBM415 structure of clinical trials based on a series of four similar structural tetrahydropyrrole derivatives were designed and synthesized, a preliminary study found that these compounds are not the same degree of bacterial PD In vitro inhibitory activity of F (IC50=2 ~ 12 nM) inhibitory activity and the proliferation of tumor cells. Objective: To evaluate the in vitro and in vivo to a series of anticancer activity, and preliminary study on its mechanism. In vitro: first we through the CCK-8 method on this subject are combined into a series of LBM415 structural analogs were determined to study the antineoplastic activity and IC50 value, finally get the compound PDF64. good activity and then we use CCK-8 method for suppressing the activity of PDF64 was measured on tumor cells in different tissues, we found that PDF64 has a broad spectrum of growth inhibition of tumor cells (IC50=2 ~ 11 M), and presents a selection of tumor cells the in vivo experiment: we use tumor model of HCT116 cells in nude mice by PDF64 in vivo antitumor activity, results showed that high dose of PDF64 in vivo showed excellent resistance Tumor effect, but there are also large.150 mg/kg PDF toxicity group average inhibition rate was 85.83%, better than the dose of compound actinonin (average positive inhibition rate 73.53%), but the toxic effects of lead in nude mice death, finally the experimental group only two nude mice survival. Immunohistochemical tumor cell proliferation in each group Ki67 activity index were found in PDF64 group the number of Ki67 positive cells was significantly lower than the control group, even lower than the Actonion group. The nude mice heart, liver, kidney, lung, analysis there is no obvious damage effect of PDF64 on the discovery and related dirty spleen stained with HE, so the toxic effects of high doses of PDF64 we need to make further discussion. Then we studied the antitumor activity of low dose PDF64, 50mg/kgPDF64 group the average inhibitory rate of tumor was 83.02% better than the dose of actinonin group (the average inhibition rate of 63.47%), the The death of mice were not found in the experimental period, body weight did not change significantly. The experimental results suggested that nude mice can tolerate 50 mg/kg PDF64 dose, and low dose of PDF64 also has a very excellent antitumor activity. The mechanism of antitumor effect: in order to investigate the mechanism of PDF64, we firstly analyze the compound effects on apoptosis of.Annexin V-FITC apoptosis PDF64 showed that apoptosis inducing effect of different degree of Jurkat and HCT116 cells, PDF64 induced apoptosis of Jurkat cells was significantly higher than that of HCT116, and has a time dose dependent. Further analysis showed that PDF64 can significantly reduce the mitochondrial membrane potential of Jurkat cells, and mitochondrial membrane potential of HCT116 cells less influence, similar PDF64 can induce the formation of ATP decreased, but the effects on Jurkat cells is more obvious. In the sign of apoptosis related protein expression The study found that Jurkat cells were treated with PDF64 after 72 h, the expression of anti apoptosis protein Bcl-2 decreased significantly, Caspase-3 was activated obviously, and detect the PARP-1 shear form. In HCT116 cells, the expression of the anti apoptotic protein Bcl-2 and did not change significantly, also did not detect the activation of Caspase3, but the detection of PARP-1 splicing forms. A series of experiments show that the PDF64 might be mediated by the mitochondrial apoptosis pathway of Jurkat cells, and the inhibitory effect of PDF64 on HCT116 cells may be associated with the other way. We use CFDA SE kit for detecting PDF64 proliferation of tumor cells, we found that PDF64 was time dose dependent inhibition of HCT116 the Jurkat cell proliferation. Cell cycle experiments, cell cycle distribution of PDF64 on Jurkat and HCT16 cells had no obvious effect on the cycle, related marker protein expression The study found that PDF64 did not make PCNA, expression of CyclinD1 and Cyclin D3 produced significant changes in.PDF64 can effectively induce Jurkat cells into the mitochondrial apoptosis pathway. Apoptosis effect on HCT116 cells is not obvious, we speculate that the effect of PDF64 on HCT116 cells may be realized through autophagy. Combined with the flow cytometry experiments by MDC staining, we found that PDF64 could effectively induce HCT116, autophagy effector Jurkat cells, and HCT116 cells autophagy induction effect is more obvious. LC3 Western blotting showed that the molecular markers of autophagy detection, by PDF64 72h, in HCT116 and Jurkat cells showed significantly increased expression of LC3- II. The experimental results PDF64,.MAPKs and AKT/mTOR induced autophagy effector HCT116 and Jurkat cells play an important role in the occurrence and development of tumor, in order to further analyze the anti-tumor effect of PDF64 We are related to mechanism, pathway activation protein and form the level of the Western blotting test. The results showed that in HCT116 cells, PDF64 inhibited p38, mTOR, Akt protein activation, whereas in Jurkat cells, PDF64 can significantly inhibit the AKT/mTOR signaling pathway in mTOR phosphorylation, and for other protein phosphorylation and non phosphorylation form has little effect. In addition PDF64 induced transcription factor c-Myc protein expression decreased with dose dependence. The existing data show that AKT/mTOR, p38 and MAPK signaling pathways and transcription factor c-Myc might be involved in tumor cell proliferation inhibition of PDF64, apoptosis and autophagy, but its specific because the mechanism of cell types and different slightly different. Conclusion: in short, four hydrogen pyrrole compound PDF64 has good anti-tumor activity in vitro and in vivo, can promote the apoptosis of Jurkat tumor cells, fine Cell proliferation and autophagy of HCT116 cells, mainly induced autophagy effect. Its anti-tumor effect may be related to AKT/mTOR pathway and c-MYC protein, but the specific mechanism and the similarities and differences in the two kinds of cells effect still need further study. The research for the design and synthesis of this kind of drugs provides the experimental basis, provides a theoretical basis for for the development of antitumor activity of PDF inhibitors.

【學(xué)位授予單位】：華東師范大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R96

【參考文獻(xiàn)】

相關(guān)期刊論文 前1條

1 Jing Deng;;How to unleash mitochondrial apoptotic blockades to kill cancers?[J];Acta Pharmaceutica Sinica B;2017年01期

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本文編號(hào)：1449125