發(fā)布時間：2018-01-20 18:13

  本文關鍵詞： 肽脫甲�；�酶 PDF64 細胞增殖 細胞凋亡 自噬 Akt/mTOR通路　出處：《華東師范大學》2017年碩士論文　論文類型：學位論文

【摘要】：背景:在過去幾十年中,細菌肽脫甲酰酶(PDF)在致病性微生物中被認為是抗菌藥物理想的潛在靶標,并且有很多的細菌PDF抑制劑被廣泛報道。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、結腸癌和肺癌等多種癌組織中高表達,其抑制劑表現(xiàn)出良好的體內外抗腫瘤活性,HsPDF有望成為極具潛力的新型抗腫瘤靶標。本課題組以目前處于臨床試驗的細菌PDF靶標化合物LBM415結構為基礎,設計合成了一系列結構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菌PDF體外抑制活性(IC50=2～12 nM)及腫瘤細胞增殖抑制活性。目的:本論文主要通過體內外實驗對這一系列化合物的抗腫瘤活性進行評價,并對其作用機制進行初步研究。體外實驗:首先我們通過CCK-8法對本課題組合成的一系列LBM415結構類似物進行抗腫瘤活性篩選及IC50值的測定,最終得到活性較好的化合物PDF64。接著我們運用CCK-8法測定PDF64對不同組織來源腫瘤細胞的抑制活性,實驗發(fā)現(xiàn)PDF64具有廣譜的抑制腫瘤細胞生長的作用(IC50=2～11μM),并呈現(xiàn)一定的腫瘤細胞選擇性。體內實驗:我們利用HCT116細胞裸鼠荷瘤模型測定了 PDF64的體內抗腫瘤活性,結果顯示,高劑量PDF64在體內表現(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%),但其毒性效應導致荷瘤裸鼠的死亡,最后實驗組僅有兩只荷瘤裸鼠存活。各組腫瘤組織細胞增殖活力指標Ki67的免疫組化檢測發(fā)現(xiàn),PDF64組Ki67陽性細胞數(shù)目明顯低于對照組,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對相關臟器的明顯損傷效應,因此高劑量PDF64的毒性效應還需要我們做進一步的探討。隨后我們又進行了低劑量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%),整個實驗周期均未出現(xiàn)小鼠的死亡,體重也沒有較明顯的變化,實驗結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。機制研究:為了探討PDF64的抗腫瘤作用機制,我們首先分析了該化合物對細胞凋亡的影響。Annexin V-FITC凋亡實驗發(fā)現(xiàn)PDF64對Jurkat和HCT116細胞有不同程度的凋亡誘導效應,PDF64對Jurkat細胞的凋亡誘導效應明顯高于HCT116,并具有時間劑量依賴性。進一步分析發(fā)現(xiàn),PDF64能明顯降低Jurkat細胞線粒體膜電位,而對HCT116細胞的線粒體膜電位影響較小,類似地PDF64能誘導ATP生成量下降,但對Jurkat細胞的作用更明顯。在凋亡相關標志蛋白表達的研究中發(fā)現(xiàn),Jurkat細胞經PDF64作用72 h后,其抗凋亡蛋白Bcl-2的表達量明顯降低,Caspase-3明顯被激活,并檢測出PARP-1剪切形式。而在HCT116細胞中,抗凋亡蛋白Bcl-2的表達量并沒有明顯變化,也沒有檢測出Caspase3的激活形式,但檢測出PARP-1剪切形式。系列實驗表明PDF64可能介導了Jurkat細胞的線粒體凋亡途徑,而PDF64對HCT116細胞的抑制效應則可能與其他的途徑有關。我們采用CFDA SE試劑盒檢測PDF64對腫瘤細胞分裂增殖的影響,實驗發(fā)現(xiàn)PDF64均能時間、劑量依賴性的抑制HCT116、Jurkat細胞的分裂增殖。細胞周期實驗發(fā)現(xiàn),PDF64對Jurkat和HCT16細胞的細胞周期分布均沒有明顯影響,在周期相關標志蛋白表達的研究中發(fā)現(xiàn),PDF64并沒有使PCNA、CyclinD1和Cyclin D3的表達量產生明顯變化。PDF64能有效地誘導Jurkat細胞進入線粒體凋亡途徑,而對HCT116細胞的凋亡效應則不明顯,我們推測PDF64對HCT116細胞作用可能是通過自噬來實現(xiàn)的。通過MDC染色結合流式細胞術實驗,我們發(fā)現(xiàn)PDF64均能有效地誘導HCT116、Jurkat細胞的自噬效應,且對HCT116細胞的自噬誘導效應更為明顯。自噬標志分子LC3 Western blotting檢測發(fā)現(xiàn),經PDF64作用72h后,在HCT116及Jurkat細胞中LC3-Ⅱ表達量明顯增加。已有的實驗結果說明,PDF64誘導了HCT116和Jurkat細胞的自噬效應。MAPKs及AKT/mTOR在腫瘤的發(fā)生發(fā)展中起重要作用,為了進一步分析PDF64的抗腫瘤作用機制,我們對相關通路的蛋白及其激活形式的水平進行了Western blotting檢測。結果顯示,在HCT116細胞中,PDF64能明顯抑制p38、mTOR、Akt等蛋白的激活,而在Jurkat細胞中,PDF64也顯著抑制了AKT/mTOR信號通路中mTOR的磷酸化水平,而對其他蛋白的磷酸化及非磷酸化形式影響較小。另外PDF64誘導了轉錄因子c-Myc蛋白表達量呈劑量依賴性的下降。已有的數(shù)據表明AKT/mTOR,p38 MAPK信號通路及轉錄因子c-Myc可能參與了PDF64引起腫瘤細胞的增殖抑制、凋亡和自噬效應,但其具體機制因細胞類型的不同又略有差異。實驗結論:總之,四氫吡咯類化合物PDF64具有良好的體內外抗腫瘤活性,對Jurkat腫瘤細胞具有促凋亡、細胞增殖抑制及自噬效應,對HCT116細胞則主要誘導自噬效應。其抗腫瘤作用可能與AKT/mTOR通路及c-MYC蛋白相關,但具體機制及在兩種細胞中效應的異同尚需進一步研究。本研究為此類藥物的設計合成提供了實驗依據,為PDF抑制劑的抗腫瘤活性研發(fā)提供了理論基礎。
[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.

【學位授予單位】：華東師范大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R96

【參考文獻】

相關期刊論文 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：1449125