【作者】 赵晓斐；

【导师】 刘相国；

【作者基本信息】 山东大学 ， 细胞生物学， 2014， 博士

【摘要】 研究背景细胞凋亡通路包括外源性死亡受体介导的信号通路和内源性线粒体信号通路。Death Receptor5(DR5)是TNFR家族的成员,包含一个细胞质内的死亡结构域(DD)。受体三聚化时,死亡结构域可以招募FADD以及pro-caspase8/10形成死亡诱导信号复合体(DISC)。随后,活化caspase并导致下游效应蛋白的切割。由于DR5对恶性肿瘤细胞有靶向选择性,很多促凋亡药物的研究都集中于DR5分子。抗凋亡蛋白c-FLIP可以阻止pro-caspase8组装入DISC进而抑制caspase8和下游效应蛋白的活化。Bcl-2家族既有抗凋亡蛋白也有促凋亡蛋白,二者的平衡对于内源性凋亡通路的调控十分重要。NOXA是一个只含BH3结构的促凋亡蛋白,是DNA损伤时p53的转录靶点。NOXA与化疗药物引起的凋亡有关,其可以与Bcl-2家族的促存活基因Mcl-1相互结合,将Bax从Mcl-1/Bax复合体中置换出来,游离的Bax进而触发内源性凋亡通路。NOXA和Mcl-1的结合也能够促进Mcl-1的蛋白酶体降解,进一步增强线粒体凋亡。很多化疗药物被报道可以诱导肿瘤细胞内质网应激(ER stress)。内质网应激多由错误折叠蛋白或未折叠蛋白在内质网腔上的聚集引起,主要包括3条信号通路,分别是蛋白激酶IRE1和PERK介导的信号通路以及转录因子ATF6介导的信号通路。错误折叠蛋白或未折叠蛋白不能降解时,内质网应激被延长并诱导凋亡。内质网应激可通过多条途径诱导凋亡,包括pro-caspasel2的切割活化、ASK1的活化等。很多研究集中在内质网应激的效应蛋白CHOP上。CHOP是ATF4的下游靶蛋白,可以作为一个含bZIP结构域的转录因子调控包括DR5和NOXA在内的凋亡相关基因。内质网应激诱导凋亡的分子机制还需要进一步的研究。肿瘤干细胞与干细胞有很多相似的特性。这类细胞具有自我更新和分化的能力,表达典型的干细胞标记物。肿瘤干细胞对药物有更强的抗性,被认为是肿瘤细胞的起源。很多报道指出肿瘤干细胞与欠佳的临床预后有关。因此,肿瘤干细胞有希望成为肿瘤治疗可行的靶点。Parthenolide (PTL)是一种来源于植物小白菊的倍半萜烯内酯。PTL因其可抑制NF-κB的活性而广泛应用与炎症的治疗。研究发现PTL还具有促进细胞分化、引起细胞周期阻滞、诱导凋亡等功能。关于其促进肿瘤细胞凋亡功能的研究发现PTL可以触发内源性线粒体信号通路,上调细胞内活性氧(ROS)水平,改变Bcl-2家族蛋白的表达。近年来有研究报道PTL可以选择性的杀伤急性骨髓性白血病干细胞和祖细胞。PTL还可以优先抑制乳腺癌类干细胞,但详细的分子机制未知。细胞内膜系统各个组分之间的物质传递常常通过膜泡运输的方式进行。膜泡运输是一种高度有组织的定向运输,马达蛋白水解ATP提供能量,使有被小泡沿着细胞内的微管被运输到靶细胞器。膜泡运输可以分为外排和内吞。其中内吞又可以根据衣被蛋白的不同分为clathrin依赖的内吞和clathrin非依赖的内吞。受体介导的内吞是细胞依靠膜表面受体特异性地摄取细胞外蛋白质或其他化合物的过程。内吞后的受体可以通过再循环途径回到质膜,也可以通过晚期内体进入溶酶体降解。内吞运输可调节多条信号通路,对细胞的极化、运动、信号传递的调控和正常细胞的生长都十分重要。不少研究认为DR5是多种不同细胞中导致死亡的首选受体。DR5也被认为是开发抗体药物治疗的首要靶点。DR5需要在膜上恰当的表达才能传递配体的凋亡信号。有研究发现,有些乳腺癌等DR5膜表面缺失的细胞系对TRAIL和受体的相关抗体有较强抗性。TRAIL处理时,TRAIL和受体DR5都迅速的内化。TRAIL能够同时通过clathrin依赖和非依赖的内吞内化。有研究发现死亡受体的活化导致了clathrin依赖的内吞中几个蛋白以caspase依赖的方式切割,抑制内吞可增强死亡受体下游caspase的活化,进一步放大了细胞凋亡但也有资料显示,有些细胞内clathrin和衔接蛋白AP2的总蛋白水平并不与细胞膜表面DR5直接相关,说明其他元件可能也参与受体的表面缺失过程。关于内吞和运输途径中导致细胞膜表面DR5缺失的分子机制需要更多的研究。这些研究对于恰当的抗肿瘤药物的选择和设计意义重大。Numb最初在果蝇中被定义为一个重要的细胞命运决定子。Numb蛋白具有调控细胞不对称分裂、决定细胞命运、内吞、细胞粘附、细胞迁移、特定底物的泛素化等功能。Numb可作为内吞蛋白,在内吞小体、clathrin有被小泡和膜泡中都有分布。内吞的全部线路中,Numb都与被内化的货物共定位,共同从中间型膜泡运输至内体。Numb可以通过与Eps15相互结合而被招募入内吞的细胞器,但Numb与Eps15不能定位在同一有被小泡,揭示了Numb可能与其他内吞机制的元件相互结合。研究方法1.肿瘤细胞培养；2. SRB和MTT比色法检测肿瘤细胞存活率；3.流式细胞术检测细胞周期4. Western Blot检测细胞内相关蛋白的表达；5.流式细胞术检测细胞凋亡；6. RNAi抑制相关基因的表达；7.基因克隆；8.流式细胞术检测膜表面DR5；9.基因过表达检测对相关蛋白的影响；10.免疫荧光检测膜表面DR5；11.免疫沉淀检测蛋白相互作用。实验结果1.PTL可明显抑制非小细胞肺癌细胞Calu-1、H1792、A549、H1299、H157和H460的存活率,这种抑制作用具有浓度依赖性；2.PTL以浓度依赖方式诱导A549细胞G0/G1细胞周期阻滞,诱导H1792细胞G2/M细胞周期阻滞；3.PTL在A549、Calu-1、H1299和H1792中以浓度和时间依赖的方式激活了促凋亡蛋白caspase的级联反应；4.PTL以浓度和时间依赖的方式促进了A549、Calu-1、H1299和H1792细胞中死亡受体DR5的表达,敲除DR5可抑制PTL诱导的caspase级联反应和细胞凋亡；5.PTL抑制了A549、Calu-1、H1299、H1792和H157细胞中抗凋亡蛋白c-FLIP的表达,这种抑制作用具有浓度和时间依赖性,过表达c-FLIP可抑制PTL诱导的caspase级联反应和细胞凋亡；6.PTL以浓度和时间依赖的方式促进了A549、Calu-1、H1299和H1792细胞中促凋亡蛋白NOXA的表达,抑制抗凋亡蛋白Mcl-1的表达,敲除NOXA可以上调Mcl-1,抑制PTL诱导的caspase级联反应和细胞凋亡；7.PTL上调内质网应激通路中的效应蛋白ATF4年CHOP的表达,这种上调具有浓度和时间依赖性,敲除ATF4抑带CHOP的表达和PTL诱导的caspase级联反应,敲除CHOP抑制了DR5和NOXA的表达,抑制了PTL诱导的caspase级联反应；8.PTL以浓度和时间依赖的方式上调了内质网应激通路中IRE1α、Bip、 p-eIF2α的表达；9.A549细胞中E-cad缺失上调干细胞标记物Oct4和Sox2表达；10.相同浓度PTL对A549/shE-cad细胞存活有更强的抑制作用,并在A549/shE-cad细胞中诱导更强的caspase级联反应；11.PTL诱导的促凋亡蛋白NOXA的上调、抗凋亡蛋白c-FLIP和Mcl-1的下调在A549/shE-cad细胞增强；12.PTL诱导的内质网应激相关蛋白p-eIF2α、ATF4和CHOP的上调在A549/shE-cad细胞增强,敲除CHOP可抑制PTL诱导的NOXA的上调以及caspase3和ARP的切割；13. SAHA以浓度梯度依赖的方式激活非小细胞肺癌细胞A549, H1792caspase级联反应；14. SAHA以浓度梯度和时间梯度依赖的方式上调A549、Calu-1、H1299和H1792细胞中DR5的表达,敲除DR5对SAHA诱导的caspase级联反应有更强的抑制作用；15.抗肿瘤药物SAHA和PEM处理可上调A549和H1792细胞膜上的DR5；16. Bafilomycin-A1和MG132可增强PEM诱导的A549细胞中DR5的表达,E-64d和MG132可增强SAHA诱导的A549和H1792细胞中DR5的表达；17. SAHA、PEM都以浓度梯度依赖的方式下调非小细胞肺癌A549和H1792细胞中Numb和NumbL的表达,18.敲除Numb和NumbL可增强抗肿瘤药物诱导的DR5表达和caspase级联反应,过表达Numb和NumbL可抑制抗肿瘤药物诱导的DR5表达和caspase级联反应；19.过表达Numb和NumbL可下调细胞膜上的DR5；20. Numb和NumbL可以与DR5相互结合。结论1.PTL具有抗肿瘤作用,其发挥抗肿瘤作用通过诱导细胞凋亡和细胞周期阻滞；2. C-FLIP和死亡受体DR5在PTL诱导的外源性凋亡中发挥重要作用；3.PTL通过NOXA-Mcl-1轴诱导内源性凋亡；4.PTL通过触发内质网应激诱导细胞凋亡；5.PTL在类肿瘤干细胞中诱导更强的内质网应激和凋亡；6.抗肿瘤药物SAHA、PEM通过上调死亡受体DR5的表达诱导非小细胞肺癌细胞凋亡；7. SAHA和PEM上调非小细胞肺癌细胞膜表面DR5,抑制溶酶体途径和蛋白酶体途径降解可增强抗肿瘤药物诱导的DR5表达；8.抗肿瘤药物通过抑制内吞蛋白Numb和NumbL上调DR5和凋亡；9.过表达Numb和NumbL减少DR5在膜上的表达；10. Numb与NumbL可以与DR5相互结合。综上所述,本研究发现PTL通过诱导细胞凋亡和周期阻滞发挥抗肿瘤作用,并且提出了PTL诱导非小细胞肺癌细胞凋亡的分子机制：PTL激活内质网应激,上调ATF4和CHOP的表达；CHOP作为转录因子上调死亡受体DR5和促凋亡蛋白NOXA的表达,从而分别诱导外源性和内源性的细胞凋亡。抗凋亡蛋白c-FLIP参与了PTL诱导的外源性细胞凋亡。本研究还发现PTL在类肿瘤细胞中诱导更强的内质网应激和凋亡,为PTL选择性杀伤作用提供了可能的分子机制。内吞蛋白Numb和NumbL可以与死亡受体DR5结合。抗肿瘤药物通过下调Numb和NumbL,上调细胞内总的和膜表面DR5,进而诱导非小细胞肺癌细胞凋亡。抑制溶酶体途径和蛋白酶体途径降解增强抗肿瘤药物诱导的DR5表达。Numb和NumbL可能通过促进DR5的内吞,下调膜表面DR5。内吞后DR5可通过溶酶体途径降解。本研究丰富了对DR5内吞和运输途径中导致细胞膜表面DR5缺失的分子机制的认知。更多还原

【Abstract】 BackgroundApoptosis consists of two major signal pathways:the extrinsic death receptor pathway and the intrinsic mitochondrial pathway. Death receptor5(DR5) is a protein that belongs to tumor necrosis factor receptor (TNFR) superfamily. It contains a cytoplasmic death domain (DD) which can recruit Fas-Associated Death Domain (FADD) and pro-caspase8/10to form the Death-Inducing Signal Complex (DISC) when the receptor is trimerized. Subsequently, initiator caspase proteins are activated to lead the cleavage of downstream effectors. Development of pro-apoptotic agonists has been focused on DR5because of its target selectivity for malignant over normal cells. The activation of caspase8and downstream effectors can be inhibited by anti-apoptosis protein c-FLIP which prevents recruitment of pro-caspase8to DISC.The imbalance among the Bcl-2family members which have been defined as either anti-apoptotic or pro-apoptotic is essential for the modulation of intrinsic apoptosis signal pathway. The BH3-only protein NOXA is a p53transcriptional target in response to DNA damage. It has been reported to be involved in chemotherapeutic agent-induced apoptosis. NOXA can interact with pro-survival Bcl-2family protein Mcl-1, results in displacing Bax from the Mcl-1/Bax complex and freeing Bax to trigger the intrinsic pathway. This combination between NOXA and Mcl-1also promote proteasomal degradation of Mcl-1to enhance the mitochondrial apoptosis.Many chemotherapy drugs have been reported to induce ER stress response in cancer cells. ER stress is usually caused by accumulation of misfolded or unfolded proteins in the ER lumen. Three signal pathways are involved in ER stress. The transducers protein kinases IRE1and PERK and the transcription factor ATF6regulate the ER stress through their respective signaling cascades. When those misfolded or unfolded proteins are not resolved, ER stress is prolonged and then induces apoptosis. There are several mechanisms linking ER stress to apoptosis such as cleavage and activation of pro-caspase12, activation of ASK1and so on. Many studies have focused on the ER stress effector CHOP, which is a downstream target of ATF4. CHOP is a bZIP-containing transcription factor that can target several apoptotic genes including DR5and NOXA. The molecular mechanisms of ER stress-induced apoptosis is remain unclear.Cancer stem cells have many similar aspects with stem cells. Those cells express typical markers of stem cells and have the ability of self-renewal and differentiation. Cancer stem cells are also considered to be the origin of cancer cells and have more resistance to anti-cancer drugs. Many reports have indicated that cancer stem cells are correlated with poor prognosis. So, targeting cancer stem cell may be a promising strategy for cancer therapy.Parthenolide is a sesquiterpene lactone derived from the plant feverfew. It has been used to treat inflammation due to its ability of inhibiting NF-κB activity. Parthenolide has also been reported to play other roles such as promoting cellular differentiation, causing cell cycle arrest and inducing apoptosis. Its pro-apoptotic effect on cancer cells is known to trigger the intrinsic apoptotic pathway by elevating levels of intracellular reactive oxygen species (ROS) and alterating of Bcl-2family proteins. What’s more, recent studies have revealed that PTL could selectively eradicate acute myelogenous leukemia stem and progenitor cells. It is also demonstrated that PTL could preferentially inhibit breast cancer stem-like cells, but the molecular mechanism is still unclear.The transfer among components of cell plasma membrane system is often executed by vesicular transport. Vesicular transport is a highly organized orientational process. With energy provided by the hydrolysis of ATP, vesicular transport makes coated pits transport to the target organelle via the microtubules.Vesicular transport includes exocytosis and endocytosis. Based on the difference of coat proteins, endocytosis can be divided into clathrin-dependent endocytosis and clathrin-independent endocytosis. Receptor-mediated endocytosis is a program by which cells ingest extracellular proteins and other compounds depended on specific receptors on the cell surface. After endocytosis, receptors can be recycled back to the plasma membrane, or transferred from the late-endosome to lysosome for degradation. Endocytosis regulates multiple signaling pathways and is important to modulate cell polarization, movement, signal transmission, and growth.Many studies considered DR5to be the pivotal death receptor in a variety of cells. DR5is also considered to be the preferred target to exploit antibodies for cancer therapy. The localization appropriacy on plasma membrane is necessary for DR5to transfer ligand signals. Studies have found that some cancer cells without surface DR5such as breast cancer cells have stronger resistance to antibodies of TRAIL and its receptors.TRAIL and its receptor DR5can be rapidly internalized after TRAIL treated. The internalization can go through both the clathrin-dependent and the clathrin-independent endocytosis. It has been reported that activation of death receptor results in cleavage of several proteins in clathrin-dependent endocytosis by a caspase-denpendent machine. Inhibition of endocytosis can enhance the activation of caspase, and enlarge cell death furthermore.However, there are also data showed that in some cells, the total protein level of clathrin and adapter protein AP2is not directly related to the surface expression of DR4and DR5on plasma membrane. Other components of clathrin-dependent endocytosis may also participate in the process by which receptors are deficiency on plasma membrane. More research about endocytosis and related molecules involved in the lack of surface TRAIL receptor is required. Those research are important for the proper selection and design of anti-cancer drug.Numb is originally defined as an important molecule that decideed cell fate in Drosophila. Numb has a variety of abilities, including the decision of cell fate, regulation of asymmetric cell division, endocytosis, cell adhesion, cell migration, specific ubiquitin of substrates and so on.Numb can act as an endocytic protein, it exists on endosome, clathrin pits and vesicles. Numb is co-located with internalized cargos, and transfer from middle vesicle to endosome through all processes of endocytosis. Numb can be recruited to endocytosis organelles by combining with Eps15, but Numb and Eps15cannot be located in a same vesicle, indicating that Numb may be combined with other components of endocytosis.Methods1. The culture of tumor cells;2. SRB and MTT assays were executed to detect the survival of tumor cells;3. Flow cytometry assay were carried out to detect the cell cycle;4. The expression level of relevant proteins in cells were detected by western blot asssays;5. Apoptosis was detected by flow cytometry assay;6. RNAi technology was used to inhibit the expression of related genes;7. Gene cloning;8. Surface expression of DR5was detected by flow cytometry assay;9. Over-expression of genes was used to assay the effects on related proteins;10. Immunofluorescence was used to test surface expression of DR5;11. Immunoprecipitation was carried out to examine the interactions between proteins.Results1. PTL can obviously inhibit the cell survival of non-small cell lung cancer cells such as Calu-1,H1792A549, H1299, H157and H460, this inhibition was in a concentration dependent manner;2. PTL induced G0/G1cell cycle arrest in A549cells in a concentration dependent manner while induced G2/M cell cycle arrest in H1792cells;3. In A549, Calu-1, H1299and H1792cells, PTL induced the activation of pro-apoptosis protein caspases in a concentration and time dependent manner;4. PTL up-regulated the expression of death receptor5in A549, Calu-1, H1299and H1792cells in a concentration and time dependent manner, knocking-down of DR5significantly inhibited the caspase cascade activation and cell apoptosis induced by PTL;5. PTL inhibited the expression of anti-apoptosis protein c-FLIP in A549, Calu-1, H1299, H1792and H157cells, this inhibition is in a concentration and time dependent manner, over-expressing of c-FLIP could significantly inhibit the caspase cascade activation and apoptosis induced by PTL;6. PTL up-regulated the expression of pro-apoptosis protein NOXA in A549, Calu-1, H1299and H1792cells in a concentration and time dependent manner, while the expression of anti-apoptosis protein Mcl-1was inhibited in an opposite manner, knocking-down of NOXA up-regulated the expression of Mcl-1and inhibited the caspase cascade activation and cell apoptosis induced by PTL7. PTL up-regulated the expression of ATF4and CHOP, which are effector proteins endoplasmic reticulum stress pathway. This up-regulation were in a concentration and time dependent manner. Knocking-down of ATF4inhibited the expression of CHOP and PTL inducing caspase cascade actication, knocking-down of CHOP inhibited the expression of DR5and NOXA and the caspase cascade activation induced by PTL;8. PTL up-regulated the expression of endoplasmic reticulum stress pathway proteins such as IRE1α, Bip, p-eIF2a in a concentration and time dependent manner;9. Deletion of E-cad increases expression of stem cell marker Oct4and Sox2in A549cells;10. The same concentration of PTL had a stronger inhibitory effect on cell survival and caspase cascade activation in A549/shE-cad cells;11. The up-regulation of pro-apoptosis protein NOXA as well as down-regulation of anti-apoptosis protein c-FLIP and Mcl-1induced by PTL were enhanced in A549/shE-cad cells;12. The up-regulation of endoplasmic reticulum stress related proteins p-eIF2a, ATF4and CHOP induced by PTL were enhanced in A549/shE-cad cells, knocking-down of CHOP inhibits the up-regulation of NOXA and the activation of caspase3and PARP induced by PTL. 13. In A549, Calu-1, H1299and H1792cells, SAHA induced the activation of caspases in a concentration dependent manner;14. SAHA up-regulated the expression of death receptor5in A549and H1792cells in a concentration dependent manner, knocking-down of DR5had a stronger effect in the inhibition of the caspase cascade activation induced by SAHA;15. Anti-cancer drugs SAHA and PEM increased surface expression of DR5in A549and H1792cells;16. Bafilomycin A1and MG132could strengthen the up-regulated expression of DR5induced by PEM in A549cells, E-64d and MG132could strengthen the up-regulated expression of DR5induced by SAHA in both A549and H1792cells;17. In A549and H1792cells, SAHA and PEM inhibited the expression of Numb and NumbL in a concentration dependent manner;18. Knocking-down of Numb and NumbL enhanced the expression of DR5and caspase cascade activation induced by SAHA and PEM; while over-expression of Numb and NumbL increased the expression of DR5and caspase cascade activation induced by SAHA and PEM;19. Numb and NumbL inhibited surface expression of DR5;20. Numb and NumbL could interact with DR5.Conclusion1. PTL has significant anti-cancer effect, which is carried out by inducing apoptosis and cell cycle arrest;2. C-FLIP and DR5play an important role in the extrinsic apoptosis induced by PTL;3. PTL induces intrinsic apoptosis through NOXA-Mcl-1axis;4. PTL induces endoplasmic reticulum stress and then trigger apoptosis;5. PTL induces stronger endoplasmic reticulum stress and apoptosis in cancer stem like cells.6. An-cancer drugs SAHA and PEM induce apoptosis in non-small cell lung cancer cells by up-regulation of DR5; 7. SAHA and PEM increase the surface expression of DR5, inhibition degradation through lysosome pathway and proteasome pathway can enhance the DR5expression induced by anti-cancer drugs;8. Anti-cancer drugs up-regulate DR5and caspase cascade activation by inhibiting Numb and NumbL;9. Numb and NumbL inhibited surface expression of DR5;10. Numb and NumbL can interact with DR5.To sum up, our study found that PTL has the ability of anti-cancer by inducing apoptosis and cell cycle arrest, we propose the molecular mechanism by which PTL inducing apoptosis in non-small cell lung cancer cells:PTL activates the endoplasmic reticulum stress, increases the expression of ATF4and CHOP; CHOP serves as a transcription factor to increase expression of pro-apoptosis proteins DR5and NOXA, which were then cause extrinsic and intrinsic apoptosis. Anti-apoptosis protein c-FLIP participates in the PTL induced extrinsic apoptosis. We also found that PTL induces stronger endoplasmic reticulum stress and apoptosis in cancer stem-like cells, which provides a possible molecular mechanism to the selective effect of PTL towards cancer stem-like cells.Endocytic protein Numb and NumbL can combine with death receptors DR5. Anti-tumor drugs can increase the total and surface expression of DR5by inhibition of Numb and NumbL, then induce apoptosis in non-small cell lung cancer cells. Inhibition of lysosome and proteasome degradation pathway can enhance the DR5expression induced by anti-cancer drugs, Numb and NumbL reduce the surface expression of DR5probably by promoting endocytosis of DR5. Our study enriches understanding about endocytosis and related molecules involved in the lack of surface TRAIL receptor.更多还原