The Mechanism Of The Key Metabolic Pathways About Peiminine On HCT-116 Cell

Objective:Colon cancer is a common malignancy in the digestive system and its incidence is rising year by year.The early and mid-stage colon cancer patients are always received surgical treatment as the first choice,and the chemotherapy regimen is determined according to the postoperative pathological stage.However,the survival and prognosis of patients with advanced colon cancer are still not optimism.Currently,comprehensive treatment for the advanced colon cancer patients is mainly based on chemotherapy,molecular targeted therapy,radiation therapy,interventional therapy and traditional Chinese medicine treatment.In China,the role of Chinese medicine in improving the quality of life of patients with cancer and improving the survival of patients with cancer has been paid attention to and developed.Many patients with advanced colon cancer tend to be benefited from integrated treatment of traditional Chinese and Western medicine.Chinese medicine treatment has some advantages in improving the quality of life and survival time of patients with colon cancer by reducing the side effects of radiotherapy and chemotherapy in patients with colon cancer,preventing postoperative recurrence and metastasis of colon cancer patients.While Chinese medicine treatment often plays a role through the treatment of traditional Chinese medicine compound,there is a large individual differences,and the specific anti-cancer mechanism and molecular targets are still not clear,so the study of anti-tumor mechanism of traditional Chinese medicine is conducive to further provide reference for clinical treatment.In previous studies,it was found that the Chinese medicine extract peiminine can inhibit the growth of human colon cancer HCT-116 cells and induce apoptosis in vitro.This study intends to further explore the effects of peiminine on key metabolic pathways of human colon cancer HCT-116 cells through metabolomics,apoptosis,autophagy research techniques,to clarify the molecular target and mechanism of peiminine on human colon cancer HCT-116 cells,and to provide an effective theoretical basis for the further development and clinical application of the peiminine.Methods:1.CCK 8 method is used to observe proliferation activity in HCT-116 cells treated by peiminine(50、100、200 and 400μM),the control group treated with the same volume of DMSO and test cell apoptosis through annexin V PI double staining and influx flow cytometry.2.GFP-LC3 plasmids are successfuliy transfected to HeLa cells.Autophagosome can be seen by confocal microscopethe postive group deal with different concentra-tions(50、100 and 200μM)of peiminine in the HeLa-GFP-LC3 cells while the control group treated with the same volume of DMSO.Through Western blotting,relative quantitation of LC3B-Ⅱ protein involved in autophagy is checked out.3.By GC-MS and UPLC/MS/MS,metabolites from the HCT-116 cells treated by peiminine and untreated have been gone through fingerprint analysis.Partial least squares discriminant(PLS-DA)analysis metabolomics data by multi-dimensional statistical.RESULT:1.We observed that peiminine treatment(50、100、200 and 400μM)decreased the number of viable HCT-116 cells in a dose-dependent manner and also significantly increased the number of annexin V-positive HCT-116 cells in a dose-dependent manner for 200 and 400μM of peiminine.This alteration is consistent with the flow cytometry assessments which showed that peiminine treatment at the same concent-rations induced early apoptosis in HCT-116 cells.2.We applied a gradient dosage of peiminine(0、50、100 and 200μM)in HeLa--GFP-LC3 cells with a control solvent and observed an increase in the GFP(green color)and lysosomes(red color)puncta in a dose-dependent manner.Immunoblot assays against LC3 B also showed a significantly elevation of the LC3B-II/ LC3B-I protein in the 50、100、200 and 400μM peiminine treated HCT-116、MEF、HeLa cells through Western blotting,especially at 200 and 400μM concentrations.3.We employed a well-established global metabolic profiling approach that combined GC/MS with UPLC/MS/MS and showed that the levels of 57 metabolites were significantly different in the groups.Of these metabolites,45 increased and 12 decreased.Particularly among the 45 increased metabolites,glutamine,glucose,andglycerol were included,which are the key molecules involved in the metabolism of acid,carbohydrates,and lipids.The remaining 12 decreased metabolites,included four amino acids,one co-factor&vitamin,four lipids,and nucleotides.Conclusions:1.Peiminine can inhibit the growth of HCT-116 cells and induce their apoptosis.2.Peiminine can enhance the expressed level of LC3B-I and LC3B-II protein in the HCT-116 cells.3.The57 markable metabolites including glucose,glutamine and lipid metab-olites upon peiminine exposure are related to the regulation of P13K/Akt/mTOR pathway and oxidative stress.