Structural Characterization of Momordicacharantia L. (Cucurbitaceae) Oligopeptides and Potency Detection in Non-small Cell Lung Cancer

Author:Dong Zuo

Supervisor:qu yi qing


Degree Year:2019





BackgroundLung cancer is one of the major causes of cancer-related death globally and the non-small cell lung cancer(NSCLC),accounting for 85%of lung cancer cases,is the most common type.Currently the available treatments for NSCLC are surgery,radiotherapy,and chemotherapy,Platinum chemotherapy is major treatment for NSCLC patients,but the effect is limited,leading to reduction of 26%-32%for death in the patients with stage Ⅲ and Ⅳ,and the median surial is still only 8 to 10 months.65%of NSCLC patients were diagnosed as advanced stage and lost operation chance.The 5-year OS is approximately 2%.Recent years the development of molecular targeted therapy had achieved great progress,for example,Gefitinib(ZD1839,Iressa),which is an EGFR tyrosine kinase inhibitor(TKI)that received approval from the FDA in 2003,inhibits EGFR activity and EGFR downstream signaling,resulting in tumour shrinkage,and patients exhibit a good initial response.However,the presence of tumor immunity reduces the anti-tumor effect and increases the dosage in the treatment.Disappointingly,large dose usage is always lead a fatal damage to the normal cells.Hence,identification of novel targets and agents foranti-NSCLCis urgent.The natural protein has been recognized for centuries with thefunctional properties.The biological activity in physiological utilization is associated with its amino acid contents.Previous studies have successfully identified that the medicinal plant provide a significant diversity of protein and peptides,which can be exploited for potential anti-cancer agents.Momordica charantia is a widely consumed vegetable with bitter taste.Furthermore,Momordicacharantia L.(Cucurbitaceae),is utilized in traditional Chinese medicine to treat a variety of diseases,such as cancer,anti-inflammation,and anti-diabetic.Momordicacharantia L.(Cucurbitaceae)extract has been demonstrated to play a role in oncogenesis,and accumulated evidence has evaluated its anticancer effects,such as anti-proliferation and anti-migration.However,to date,no work focused on the Momordica charantia L.(Cucurbitaceae)oligopeptides(MCLOs)for cancer therapy.In this study,a serious of MCLOs were isolated and the amino acid sequence were identified,the anticancer activities against NSCLC were evaluated,and the related mechanisms were also invested.ObjectiveIn current study,we isolated a series of MCLOs using a line of purification techniques and identified their amino acid sequence.We further investigated its anti-tumor effects against NSCLC cells:A549,L78,PGCL3,H460 and NCI-H1299 cells and potential mechanism,thus clarify their anti-tumor effects.Methods1.Isolation and Identification of MCLOs:The crude extracts(Fraction I)were obtained by ammonium sulfate precipitation(50%-80%saturation),ultra-filtration(1kDa cut-off)and lyophilization.Further,the fractions with anti-cancer potency were purified using Hydrophobic Chromatography,Anion-exchange Chromatography,Gel filtration Chromatography and RP-HPLC.During the separation and purification process,the protein elution profile was monitored at 280 nm and MTT assay was performed to monitor the anti-tumor activity.2.We used MTT method to detect the proliferation of A549,L78,PGCL3,H460,NCI-H1299 and normal lung fibroblast CCD 19cell lines3.Annexin V/PI detection kit and flow cytometry were used to detect the effect of MCLO-12 in inducing apoptosis and arresting cell cycle in A549 cells.4.A549 cells were incubated with different concentration of MCLO-12 for 48h;JC-1 and ROS assay kits were used to detect the mitochondrial potential and intracellular ROS level.Western blotting assay was used to detect related protein expression.Results1.In this study,twenty-eight MCLOs were isolated,and amino acid sequences identified by HPLC-ESI-MS.MCLO-12 was determined to be 811.3761Da,the sequence is FHGKGHE2.Most of the MCLOs performed anti-proliferative activities,but not on human normal lung fibroblast cells.MTT cell proliferation assay showed that the proliferation of NSCLC cells were significantly inhibited after the treatment with different concentrations of MCLOs for 48h.MCLO-12 showed the best anti-proliferation activity in the A549 cells with an IC50 value of 21.4±2.21 mM,while the IC50against the human lung fibroblast cell line CDD19 cells is>200 mM3.MCLO-12 induced cell apoptosis,especially late apoptosis in a dose-depend dose-dependent manner.MCLO-12induced cell apoptosis by blocking the cell cycle at G0/G1 phase in A549 cells4.MCLO-12 increases cellular ROS level and induces MMP Loss:MCLO-12 caused a clearly ROS accumulation in a dose and time-dependent manner.MCLO-12 down-regulated mitochondrial membrane potential in A549 cells.Further,the cells incubated with 42.8 mM MCLO-12 was almost completely lost5.Western blotting assay showed that MCLO-12 induced cell apoptosis by regulating Caspase 3,Caspase 9 and PARP expressions,which closely involved in the program of cell apoptosis.Further,MCLO-12 induced cell apoptosis by Suppressing Trx system and activating ASK1 and MAPK pathways.Conclusion1.MCLO-12 had a selective inhibition effect in NSCLC cells,and it could induce the cell apoptosis and cell cycle arresting.2.The ability of MCLO-12 to inhibit the proliferation of A549 cells by inducing apoptosis via mitochondrial pathways.Suppression of the Trx system with MCLO-12 could lead an ROS accumulation and subsequently activate a number of Trx-dependent pathways,including the ASK1,MAPK-p38 and JNK pathways,which may lead to MCLO-12-mediated apoptosis.