Annonaceous Acetogenins-mediated Up-regulation Of Notch2 Signaling Exerts Cell Growth Inhibition In Human Gastric Cancer Cells

BackgroundGastric cancer (GC) is one of the most common malignancies and one of the most important causes of cancer-related death worldwide. Most of current available therapeutic methods for gastric cancer like chemotherapy and chemoembolization, are less optimal, thus the prognosis of GC is rather poor. This is largely attributed to a lack of complete understanding of the mechanisms for this malignancy. Therefore, continued exploration of alternative treatment options is critical to developing more effective therapies.One promising candidate for chemotherapeutic and chemopreventive drugs is traditional Chinese medicines. A variety of novel natural compounds, such as paclitaxel, podophyllotoxin, vinblastine and camptothecin, have been isolated and used as agents for cancer chemotherapy and chemoprevention. Annonaceae, also called custard apple family or soursop family, with approximately 120 genera and 2,000-2,200 species, is the largest family in Magnoliales. The whole plants, fruits or seeds of Annonaceous plants have been widely used in traditional and popular medicines for treating pain, fever, diarrhea, hypotension, internal, and external parasites. Annonaceous acetogenins (ACGs), a group of fatty acid derivatives, are proprietarily isolated from Annonaceae plants. When the first ACG compound, uvaricin, was isolated in 1982, its excellent in-vivo anti-leukemia effect aroused widespread interest of natural product and medicinal chemists to isolate and identify this class of compounds. ACGs exhibited a broad spectrum of biological properties, such as cytotoxicity, anti-tumoral, anti-malarial, anti-microbial, anti-viral, anti-feedant, anti-helminitic, pesticidal, and immunosuppressive activities, suggesting many potentially useful applications. The powerful cytotoxicity and anti-tumor activity made ACGs as another "tomorrow anticancer star" after paclitaxel.Notch signaling, as an evolutionarily conserved signaling pathway, is involved in a variety of cellular processes including cell fate, differentiation, proliferation, survival rate, and apoptosis. Additionally, dysregulation of Notch signaling is associated with oncogenesis, which usually manifests as abnormality of Notch signal components like ligands, receptors, and downstream proteins. The Notch signaling in mammals consists of four receptors (Notch 1-4) and five ligands [Delta-like ligand 1/3/4 (DLL1/3/4) and Jagged (1/2)]. After binding of the receptors to their ligands, the y-secretase complex mediates the cleavage of the transmembrane domain of Notch receptor to release the intracellular domain of Notch receptor (NICD). Then, NICD translocates into the nucleus and works as a transcriptional coactivator, thus regulating the expression of target genes, including the hairy enhancer of split (Hes) and Hes-related (Hey) family. Clearly, Activated Notch receptor can be oncogenic or tumor suppressive depending on the tumor type and cellular context. For instance, Notch has an oncogenic role in colorectal cancer, breast cancer, lung cancer, and neuroblastoma. On the contrary, Notch acts as a tumor suppressor in squamous cell carcinoma of the skin and cervical uterus, hepatocellular carcinoma and neuroendocrine tumors of the lung and gastrointestinal tract. Currently, a number of studies have been conducted to investigate the association between Notch signaling pathway and GC in humans. However, the function of components of Notch pathway in GC is still controversial because different even opposite effects have been indicated. Notch1 has been found to be expressed in most GC cell lines as well as normal gastric mucosa, but other data demonstrated that no expression was detected in normal gastric mucosa. The meta-analysis by Du X et al. revealed that Notch 1 was expressed in both GC tissues and normal mucosa, but significantly higher expression was seen in cancer tissues than in normal tissues, suggesting that Notch 1 is activated in GC. This is consistent with the role of Notch1 as an oncogene in many solid malignancies. As for Notch2, Sun et al. proved that Notch2 was overexpressed in GC. More importantly, co-expression and nuclear co-translocation of Notch2 and downstream protein Hes1 were found to be more frequent than Notch 1 both in vivo and in vitro in gastric cancer, suggesting that Notch2 mediated signaling would be more important in GC carcinogenesis and progression. Tseng et al. showed that activation of Notch2 would promote both cell proliferation and xenografted tumor growth of GC cells through cyclooxygenase-2. Conversely, Guo et al showed that Notch2 as a tumor suppressor gene could negatively regulate cell invasion of human gastric carcinoma. Therefore, the multifaceted features of Notch family members suggest the necessity to check the activation patterns and potential roles of Notch signaling in different tumor types without any initial impression.Up to date now, the role of Notch2 singling in the antitumor activity of ACGs has not been examined. Among them, the Notch2 intracellular domain (N2ICD) plays a role in intracellular signal transduction. In this study, ACGs was administered in GC cells to determine the cellular processes affected by this compound and whether it played a tumorsuppressor role through the regulation of Notch2.This project is aimed to study the effects of ACGs on gastric cancer cell lines AGS and MKN-45. The details are as follow:1. Eukaryotic expression of N2ICD:To construct recombinant eukaryotic expression plasmid N2ICD/pCMV-Tag4 and transfect it into HEK 293T cell. The recombinant eukaryotic plasmid N2ICD/pCMV-Tag4 will be used for N2ICD overexpression in our following research.2. Prokaryotic expression of N21CD and purification:To construct recombinant prokaryotic plasmid N2ICD/pGEX-4T-1 and express it in E.coli BL21. The purified N2ICD will be used in pull-down assay to identify proteins interacted with N2ICD.3. Annonaceous acetogenins-mediated up-regulation of Notch2 signaling exerts cell growth inhibition in human:The effect of ACGs on cells’ proliferation, apoptosis,cell cycle and the roles of Notch2 pathway on the activity of ACGs were assessed. Furthermore,Notch2 overexpression and Notch2-siRNA interference were performed to investigate the effect of ACGs on GC cell proliferation. This study will provide a new strategy on GC therapy.PurposesTo explore whether ACGs inhibits the growth of gastric cancer cell throught Notch2 signal pathway.Methods1、Human Notch2 intracellular domain (N2ICD)gene was amplified from Hela cell by RT-PCR, identified by enzyme restriction, sequencing and blasting and then cloned into eukaryotic expression vector pCMV-Tag4 to construct recombinant eukaryotic plasmid N2ICD/pCMV-Tag4, which was then transiently transfected into HEK 293T cell. Q-PCR and Western Blotting were perfomred to detect the expression of target protein.2、N2ICD was amplified via PCR from recombinant eukaryotic plasmid N2ICD/pCMV-Tag4, identified by enzyme restriction and sequencing and then cloned into prokaryotic expression vector pGEX-4T-l carrying glutathione S-transferase (GST) tag to construct recombinant prokaryotic plasmid N2ICD/pCMV-Tag4. Then the recombinant plasmid was transformed into E.coli BL21 and induced by Isopropyl β-D-Thiogalactoside (IPTG). After the bacterial sediment was lysed by repeating freezing and thawing, lysozyme lysis and ultrasonication, the supernatant was purified by Glutathione Sepharose 4B and Western Blot were carried out to confirm the purified product.3、The effects of ACGs on GC cell proliferation were assessed by MTS. Additioally, The effects of ACGs on ACGs cell apoptosis and cell cycle were identified by flow cytometry. Then the expression of Notch2 and its downdream protein Hesl after ACGs treatment was confirmed by Western Blot. The roles of Notch2 were further proved by over-expression of Notch2-cDNA transfection and down-regulation of Notch2-siRNA prior to ACGs administration.ResultsN2ICD gene was successfully amplified from Hela cells without any gene mutation and cloned into pCMV-Tag4 to successfully construct recombinant eukaryotic plasmid N2ICD/pCMV-Tag4. The recombinant plasmid recombinant plasmid, which was transfected into HEK 293T cell and succeffuly expressed.N2ICD was obtained using N2ICD/pCMV-Tag4 as template via PCR and cloned into pGEX-4T-1 to successfully construct recombinant prokaryotic plasmid N2ICD/pGEX-4T-1. The recombinant plasmid was transfected into E.coli BL21 and expressed after IPTG induction. Target protein was expressed in both supernatant ans precipitation.The supernatant was purified by Glutathione Sepharose 4B. Western Bloting confirmed that fusion protein GST-N2ICD was successfully expressed and purified.ACGs exhibited great cytotoxicity on GC cells and resulted in dose and time-dependent inhibition of cell viability as well as the induction of apoptosis demonstrated by MTS assay and flowcytometry analysis, respectively. Additionally, cell cycle analysis revealed that ACGs induced a dose-dependent accumulation of GC cells in G0/G1 phase. Then the role of Notch2 in ACGs’activities was explored. The results revealed that ACGs treatment up-regulated Notch2 expression in a dose-dependent manner in GC cells, suggesting Notch2 acted as a tumor suppressor. The over-expression of Notch2 by cDNA transfection promoted ACGs-induced cytotoxity to a certain degree compared with ACGs alone. Conversely, the knockdown of Notch2 by siRNA, together with ACGs treatment, reduced the inhibition of cell growth to a greater degree compared with ACGs alone.ConclusionACGs administration in GC cells in vitro resulted in increased inhibition of cell proliferation, apoptosis, G0/G1 arrest and Notch2 expression in a dose-dependent manner. Furthermore, overexpression of Notch2 increased ACGs cytotoxity and Notch2-siRNA reduced ACGs cytotoxity. Therefore, ACGs might exert its antitumor effects through directly or indirectly activating Notch2 signaling pathway.