| BACKGROUND AND OBJECTIVEAutophagy is a tightly regulated intracellular process through which cytosolic components, ranging from proteins, lipids, sugars and nucleotides to whole organelles and invading pathogens, are targeted for lysosomal degradation. The degradation products can be reused by cells for nutrients recycling and organelles turnover. So autophagy has proved to be important in the maintenance of cellular homeostasis and of the energetic balance. It can be stimulated by multiple forms of cellular stress, including nutrient or growth factor deprivation, hypoxia. reactive oxygen species, DNA damage, protein aggregates, damaged organelles, or intracellular pathogens. And in this situation autophagy mainly plays an adaptive role in protecting organisms against a variety of pathologies. Howerver, disregulation of autophagy is closely associated with various diseases such as heart disease, neurodegenerative disease, cancer, atherosclerosis, and aging. Thus, exploiting the molecular mechanisms of autophagy may undoubtedly have important pathophysiological and physiological implications.Vascular endothelial cells (VECs) are a simple monolayer of cells covering the entire inner surface of all the blood vessels. It has recently been established as a strategically-located multifunctional organ and plays many pivotal roles in the regulation of vascular tone and endothelial integrity as well as in the maintenance of blood fluidity and homeostasis. Endothelial dysregulation may develop secondary to hypertension, atherosclerosis, hyperglycaemia or others. Both autophagy and endothelial dysregulation has a relationgship with some diseases. Therefore, the role of autophagy and its modulation in normal endothelial functions became a research hotspot in recent years. In our previous study we have found that chemical small molecule ABO could stimulate autophagy in human umbilical vein endothelial cells (HUVECs) and protect it from apoptosis in the absence of bFGF-2and serum. For further investigation we found that ANXA7was essential for autophagy induction via modulating the intracellular calcium concentration evoked by ABO, and different from the pathway of mTOR and ROS, which indicates that ANXA7might induce a new autophgic pathway, yet the downstream factors are not clear. Annexin A7(ANXA7, also called synexin) is a member of the class of Ca2+-dependent, negatively charged phospholipid-binding proteins, plays a role in regulating intracellular calcium homeostasis and forming a Ca2+channel.ANXA7is important for autophagy of vascular endothelial cell. However, the underlying mechanism remains largely unknown. Here, we use the yeast two-hybrid system to screen and identify the interacting protein with ANXA7and to study the relationship of the interaction and the autophagy regulation, and provide evidence for elucidating the molecular mechanism of autophagy in HUVECs. Small molecules that temporarily regulate protein function and affect biological processes in cells and organisms have been extensively employed to dissect biological pathways and to study diseases, and lay a basis for the development of treatment related diseases drugs. Besides, it has a closely association between autopahgy and apoptosis, yet the molecular links are poorly understood. The purpose of our study that screening and identification chemical small molecules that can regulate lung cancer cells autophagy and apoptosis, was to provide new tools and pointcut for further investigating the complex molecular mechanisms underlying autophagy and apoptosis of lung cancer cells, and provide pilot compounds for developing treatment drugs.STUDY CONTENTS1. Screening and identification of proteins interacting with ANXA7by yeast two hybrid system(1) Construct the bait expression vector of NpGBKT7-ANXA7(2) Test of the toxicity effect and autonomous activation of NpGBKT7-ANXA7plasmid(3) Screening of the human liver cDNA library (4) Analyse the positive colonies plasmids with bioinformatics, and verified the interaction between the candidate proteins and ANXA7in VECs.2. Study the role of the grancalcin autophagy in ABO-induced VECs autophagy.(1) Effect of ABO on the interaction of grancalcin and ANXA7(2) Effect of ABO on the distribution of grancalcin in VECs(3) Effect of grancalcin on the autophagy of VECs evoked by ABO(4) Effect of the interaction between grancalcin and ANXA7on the Ca2+of VECs3. Screening and action mechanism study of cancer cells autophagy and apoptosis inducer.(1) Effects of compounds on the biological activity of wide-type p53A549cells(2) Effects of compounds on the biological activity of mutant p53H322cells(3) Effects of compounds on the biological activity of null p53H1299cells(4) Effects of compounds on the growth of normal human embryo lung cells.METHODSI. Screening and identification of proteins interacting with ANXA7by yeasttwo hybrid system and research the role of autophagy in HUVECs.1. Vector construction (referred to Molecular Cloning):The yeast expression vector NpGBKT7(derived from pGBKT7) was used to express the ANXA7fusion proteins. The full length of human ANXA7cDNA was amplified by PCR from me pCMV6-XL5-ANXA5with primers ANXA7-F/R, and two restriction enzyme sites Sal I/Not I were introduced into by PCR according to the ORF of the yeast expression vector NpGBKT7. After being digested the PCR products were ligated into NpGBKT7.2. Test of the toxicity effect and autonomous activation of NpGBKT7-ANXA7plasmid:NpGBKT7-ANXA7was transformed into competent S.cerevisiae AH109and the positive clones on the plate of SD/-Trp were screened according to Yeast Transformation System2manual book (Clontech). The toxicity effect was testified by phenotype and optical density. And autonomous activation was determined by transform both plasmids NpGBKT7-ANXA7and pGADT7into AH109on the plates of SD/-His/-Trp/-Leu with different concentrations of 3-AT.3. Screen of the cDNA library:The human cDNA library was transformed into competent S.cerevisiae AH109containing the bait plasmid NpGBKT7-ANXA7according to clontech manufacturer’s protocol. Spread100100μl serial dilutions onto a φ9cm plate containing the appropriate SD/-Trp/-Leu for determination of transformation efficiency, the resultant plate onto the SD/-His/-Trp/-Leu medium with2.5mM3-AT.4. Cell culture and transfenction:Cos-7cells were grown in DMEM with10%FCS, penicillin (100Units/ml) and streptomycin (100mg/ml). The expression vectors were transfected into Cos-7using LipofectamineTM2000reagent (Invitrogen) according to the manufacturer’s protocol.5. Verify the protein-protein interactions:Co-immunoprecipitation and Confocal microscopy6. The isolation and culture of HUVECs:The extraction and culture of HUVECs were carried out according to Jaffe et al (Jaffe et al.,1973).7. Analysis of expression and distribution of proteins:The intracellualr distribution of grancalcin was analyzed by immunocytochemistry. The protein levels were evaluated by western blot assay.8.RNA interference (RNAi):Specific siRNAs against Grancalcin were purchased from santa cruze. Cells at80-90%confluence were transfected with scrambled RNA (negative control) or siRNA against Grancalcin by RNAiFect Transfection Reagent according to the manufacturer’s instructions (QIAGEN,1022076). Western blot analysis was used to estimate the effect of gene silencing.9. Measurement of cytosolic free calcium:The fluorescence probe Fluo-3AM was utilized to measure the cytosolic Ca2+. Data were analyzed with Leica Confocal Software (LCS Lite).II. Screening and action mechanism study of cancer cells growth inhibition, autophagy and apoptosis inducer1. The morphological changes were observed under a phase contrast microscope.2. The cell viability was measured by SRB assay. 3. LDH assay was performed to determine whether cells treated with small molecules underwent necrosis.4. To investigate whether compounds induce autophagy, acridine orange (AO) staining and autophagy-related proteins of LC3-Ⅱand p62were analyzed by under a fluorescent microscope and western blot assay respectively.5. Hoechst33258staining was used to detect whether compounds induce apoptosis.RESULTSI. Screening and identification of proteins interacting with ANXA7by yeast two hybrid system and research the role of autophagy in HUVEC.1. Identification four proteins interacting with ANXA7by yeast two hybrid system, which is grancalcin, TIA1cytotoxic granule-associated, RNA binding protein, fibrinogen beta chain and complement factor H1.1Cloning and sequence analysis of ANXA7transcript:The open reading frame of ANXA7is composed of1467nt. Recombinant plasmid NpGBKT7-ANXA7was identified to be constructed correctly after being testified by colony PCR, digestion and sequencing (FigureⅠ-1-2,Ⅰ-1-3).1.2The toxicity effect and autonomous activation of NpGBKT7-ANXA7:The DNA-BD/bait fusion protein expressed by the recombinant bait plasmid NpGBKT7-ANXA7is nontoxic to yeast train AH109(Figure Ⅰ-1-5). and2.5mM3-AT is sufficient to suppress leaky HIS reporter genes (Figure1-1-6).1.3Screen of the cDNA library:1.72×106transformants were screened (Figure Ⅰ-2-1). After four rounds screening of the cDNA library, four positive clones were identified (Figure Ⅰ-2-2,1-2-3,Ⅰ-2-4,Ⅰ-2-5).1.4Analysis of positive target clones by bioinformatics:Based on the aligned result, the resource and function of proteins which are encoded by the positive recombinant library plasmids were analyzed and acknowledged false positive protein were deleted, i.e, mitochondrial protein (haplotype As SD mitochondrion), ferritin and cell skeleton (actin). Four positive target proteins were identified: grancalcin, TIA1cytotoxic granule-associated RNA binding protein, fibrinogen beta chain, complement factor H (table1)2. Verify the interaction of ANXA7with GCA, TIA-1, CFH and FGB in cells.2.1The encoding sequence of GCA, TIA-1, CFH and FGB were from human liver cDNA library. cDNA was PCR amplified and cloned into pEGFP-C2and pMyc-C2respectively. After being digested by endonucleases and sequencing, confirm vector construction success (FigureⅠ-3-1,Ⅰ-3-2). The expression vectors were transfected into Cos7cells, fluorescence imaging and western blot analysis show fusion protein expression.2.2pEGFP-ANAX7was cotransfected into Cos7cell with pMyc-GCA, pMyc-TIA-1, pMyc-CFH, pMyc-FGB. Empty pMyc-C2used as control.48h after transfection, cells were lysed on ice-cold buffer and IP were carried out use mouse anti-Myc monoclonal antibody. A GFP rabbit mAb was used to recognize GFP-ANXA7. a protein of74kDa was detected in immunocomplexes corresponding to molecular weight of GFP and ANXA7in Cos7cells but not with control. The results showed that GFP-tagged ANXA7interact with Myc-tagged GCA. TIA-1, CFH and FGB fusion protein (Figure Ⅰ-3-6).The interaction between ANXA7and TIA-1, FGB were further validated by reverse immunoprecipitation (Figure Ⅰ-3-7),2.3GCA, TIA-1. CFH and FGB antibody were used as immunoprecipate antibody, A ANXA7rabbit mAb was used to recognize ANXA7.As shown in Figure Ⅰ-3-8. ANXA7was detected in immunocomplexes against GCA, TIA-1, FGB, which indicate ANXA7with GCA, TIA-1. FGB but not CFH in HUVECs.3. Grancalcin plays a crucial role in autophagy evoked by ABO.3.1The interaction of ANXA7and grancalcin was enhanced treated with ABO: To investigate the effect of ABO on the interaction between ANXA7and grancalcin, HUVECs were treated with R-ABO and S-ABO for6h,12h or24h, and then equal amount of protein incubated with anti-grancalcin IgG. The protein level of ANXA7was elevated evidently (Figure Ⅰ-4-2A). Aliqute of cell lysates were incubated with R-ABO and S-ABO, or ABO(mixture R-ABO and S-ABO) in the presence of anti-grancalcin IgG. the results indicate that three types of ABO promote ANXA7binding to grancalcin (Figure Ⅰ-4-2B). 3.2ABO regulated distribution of grancalcin:After HUVECs were treated with ABO for24h, cells were fixed with4%paraformaldehyde for immunostaining assay. The results showed that the ABO-treated cells exhibited higher fluorescence intensity of gracalcin per cell in a noticeable punctate pattern (Figure Ⅰ-4-3).3.3GCA silencing downregulate the protein level of GCA:20nM and40nM siGCA were transfected into HUVECs. Transfected cells were harvested at24hours and48h after transfection. Western blot analysis show expression of grancalcin was efficiently reduced at concentration of40nm (Figure Ⅰ-4-4).3.4Grancalcin participated in the autophagy induced by ABO:In order to evaluate whether grancalcin play a role in autophagy. HUVECs were incubated with scrambled RNA and siRNA against grancalcin for24h, and then treated with ABO. The results showed that ABO could not induce autophagy any more according to detect the accumulation and distribution of LC3compared with Scr (Figure1-4-5).3.5GCA silencing did not affect ANXA7protein level:Cells were transfected with40nm siGCA, with scrambled RNA as control.and then ABO was added. After24h transfection, total cell lysates were harvested and detected using western blot.The results showed that ANXA7level was increased evidently (Figure1-4-6), in accord with the previous study.3.6Grancalcin influence the level of Ca2+:To evaluate the role of grancalcin in Ca2+control, we knockdown the grancalcin in endothelial cells. The data show the Ca2+of the cell dramatically elevated transfected with siRNA against grancalicn than control (Figure Ⅰ-4-7).Ⅱ. Screening and action mechanism study of cancer cells growth inhibition, autophagy and apoptosis inducer1. Effects of pyrazolo[1,5-a]pyrazin-4(5H)-one derivatives(3a-31) on the biological activity of A549cells.1.13a-31(structure:Figure Ⅱ-1)do not affect the morphology:A549cells were treated with twelve pyrazolo [1,5-a] pyrazin-4(5H)-one derivatives for24h and 48h at the concentration of8μM,16μM,32μM and64μM. Compared with the control, A549cell morphology treated with compounds has not obviously changed, only the cell number decreased at high dose treated with3e-3h (Figure Ⅱ-2A,Ⅱ-2B).1.23e-3h inhibit the proliferation:A549cells treated as described above. The SRB results showed3e-3h could significantly inhibit the proliferation at32μM and64μM(Figure Ⅱ-3).1.33e-3h increase the acidic vesicles:A549cells were treated with3e-3h for24h and48h, and then visualized by acridine orange staining. Compared with control, the acidic vesicles accumulated significantly after treatment. The results showed that3e-3h may induce A549cells autophagy (Figure Ⅱ-4). To further determine whether3e-3h induce autophagy. we detected the accumulation of LC3-Ⅱ and degradation of p62by western blot. Protein level of LC3-Ⅱ and p62has no significant difference with control,which indicate3e-3h did not induce antophagy of A549cells (Figure Ⅱ-5).1.43e-3h did not induce necrosis:LDH assay showed that the four compounds did not induce necrosis (Figure Ⅱ-6).1.53e-3h did not induce apoptosis:Hoechst33258staining showed no characteristics of apoptotic cells such as DNA fragmentation, chromatin condensation, cell shrinkage, and membrane blebbing was observed, indicate that3e-3h did not induce apoptosis in A549cells (Figure Ⅱ-7).1.63e-3h arrest the cell cycle:To determine the mechanisms of3e-3h inhibit proliferation of A549cells, the cell cycle progression was investigated by Flow cytometry analysis. The results showed3e-3h induced a strong G1-phase arrest (Figure11-8).2. Effects of3e-3h on the biological activity of H322ans H1299cells.2.1H322and H1299cells were treated with3e-3h at the concentration of16μM,32μM and64μM. Cell shrinkage, and membrane blebbing were observed by optical microscope (Figure Ⅱ-9A, B; Ⅱ-10A, B).2.2SRB assay showed cell viability of H322and H1299cells treated with3e-3h reduced siginificantly (Figure II-9C, II-10C).2.3Hoechst33258staining showed chromatin condensation and DNA fragmentation in the cells were determined by fluorescence microscope, which indicate3e-3h induce apoptosis in H322and H1299cells (Figure II-9D, E; Ⅱ-10D, E).3. Effects of3e-3h on normal human embryo lung cells. We further investigate effects of3e-3h on normal human embryo lung cells. Cells were treated with compounds3e-3h,5-FU (positive control) or0.1%DMSO (vehicle control) for48h at the concentration of32and64μM. SRB assay showed3e-3h had no effects on the proliferation of A549cells (Figure II-11).CONCLUSIONSI. Identified four proteins that can interact with ANXA71. Recombinant plasmid NpGBKT7-ANXA7was identified to be constructed correctly.2. The DNA-BD/bait fusion protein expressed by the recombinant bait plasmid NpGBKT7-ANXA7is nontoxic to yeast train AH109, and2.5mM3-AT is sufficient to suppress leaky HIS reporter genes.3. Screen of the cDNA library and indentified four candidate proteins interact with ANXA7:grancalcin. TIA1cytotoxic granule-associated RNA binding protein, fibrinogen beta chain, complement factor H.4. Grancalcin, TIA1cytotoxic granule-associated, RNA binding protein, fibrinogen beta chain can interact with ANXA7in HUVECs.5. Grancalcin is essential for the autophagy of HUVECs evoked by ABO. We found ABO could enhanced the interaction of ANXA7and grancalcin, while downregulating the protein level of grancalin, ABO could not induce autophagy and the cytosolic Ca2+of the cells dramatically elevated.Ⅱ. In this part, we found four (3e-3h) of the12compounds can inhibit the growth of Lung adenocarcinoma cells efficiently.3e-3h inhibited growth of A549cells by inducing a strong G1-phase arrest, whereas these compounds inhibited growth of H1299and H322cells by inducing apoptosis. |
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