Evodiamine Activates Autophagy Via AMPK/mTOR Pathway To Inhibit The Proliferation In Colon Cancer Cell

Colon cancer is the most common gastrointestinal cancer worldwide.Despite great achievements in surgery,chemotherapy,and the development of novel molecular-targeted drugs,the incidence of colon cancer continues to increase.Early diagnostic and therapeutic strategies can improve the overall survival of patients with colon cancer.Currently,wide use of adjacent chemotherapy following surgery resection has dramatically improved the clinical outcome of colon cancer patients.However,for patients diagnosed with advanced-stage disease,colon cancer has often metastasized.The majority of these deaths are caused by the progression of the tumor to metastatic disease.The 5-year survival rate for metastatic colon cancer is 10–15%,compared to 40–90%for non-metastatic colon cancer.Early screening and novel therapeutic approaches are essential for improved prognosis of colon cancer.Herbal medicines such as Evodiamine have long been accepted as useful adjacent therapeutic regimens for various diseases.In recent years,many studies have reported anti-cancer effects in different cancer types,including breast cancer and oral cavity cancer.However,the effects of evodiamine in human colorectal cancer cells and the underlying molecular mechanisms have been poorly determined.Autophagy,type II programmed cell death,is characterized by the degradation of cellular components including Golgi apparatus,mitochondria,polyribosomes and endoplasmic reticulum as well as the formation of numerous autophagosomes.Autophagy is activated in response to stressful stimuli,including starvation,hypoxia and high temperature or intracellular stress such as damaged organelles and mutant proteins.During the cellular process of autophagy,the redundant,damaged or aged organelles and cells are sequestered,degraded and recycled.One important function of autophagy is to overcome stress conditions and maintain cellular homeostasis.Impaired autophagy is also involved in various diseases including neurodegeneration,cardiovascular diseases,autoimmune diseases,aging,rheumatoid arthritis,infection and cancers.Currently,the role of autophagy in tumorigenesis has attracted much attention.The death-promoting effect of autophagy may lead to growth inhibition of cancer cells and suppress tumorigenesis.AMPK?adenosine monophosphate-activated protein kinase?is one of the main cellular sensors able to link a variety of cellular functions and processes to energy availability.AMPK is an evolutionarily conserved protein kinase,present both in unicellular organisms,such as baker's yeast,and also in more complex multicellular eukaryotes,as mammals.AMPK signaling pathways are involved in numerous physiological processes apart from their main metabolic functions,such as cytoskeleton remodeling and transcriptional control or regulation of essential cellular processes,such as apoptosis or autophagy.Studies have shown that Evo can activate AMPK,thus exerting biological functions.Therefore,this study proposes the hypothesis that Evo inhibits colon cancer cell proliferation in vitro and in vivo by activating AMPK signaling pathway and activating autophagy.MethodsIn vitro:Human colorectal cancer cell lines HCT–16 was cultured for24h in vitro,then cocultrued with Evodiamine at the concentration of 0,1.5,3.0,6.0?mol·L^-1 for 48h,respectively.The proliferation of cells was detected by CCK-8,Cell clone formation assay and Edu staining;Cell autophagy was detected by MDC,and RFP-GFP-LC3 adenovirus;The ROS was detected by DHE.Cells treated with 0,3,6?mol·L^-1 Evo for 48h,and then analyzed the protein and gene levels of LC3,p62,AMPK,p-AMPK,mTOR,p-mTOR by RT-PCR and western blot.Evo?6?mol/L?was co-operated with autophagy inhibitor 3-Methyladenine and apoptosis inhibitor Z-DEVD-FMK for 48 h,autophagy and apoptosis-related proteins were detected by Western blot.In vivo:Balb/c nude mice were randomized into two groups.Each mouse was inoculated subcutaneously in the right lower quadrant with 1×10⁷ HCT-116 cell.After the diameter of the tumor grew to about 0.5 cm,Evo?10mg/kg?were given to the tumor-bearing mice.We record the weight of mice in each group and volume,diameter,weight of tumor.HE coloration was used to observed cell morphology.The expression of LC3?p62?AMPK?p-AMPK?mTOR?p-mTOR proteins were determined by western blot and Immunohistochemical staining.Results1.HCT-116 cells were treated with different concentration of Evodiamine for 48 h,the proliferation of cells was significantly inhibited by Evodiamine,in a dose-dependent manner.At the same time,it can obviously reduce the formation of cell clone.After administration of the apoptosis inhibitor Z-DEVD-FMK,the cell proliferation and colony formation were further inhibited.2.Evo significantly increased the production of autophagy and ROS,upregulated the ratio of LC3 and downregulated the expression of p62 in HCT-116 cells,and upregulated the expression of key proteins in AMPK/mTOR signaling pathway.When an autophagy inhibitor3-Methyladenine was administered,the apoptotic signaling pathway was further activated,whereas when an apoptosis inhibitor Z-DEVD-FMK was administered,the autophagy signal was further activated.3.In vivo experiment,Weight of tumor in Evo group was significantly lower than that of control group?0.71±0.22g VS 3.51±0.6g?.Volume of tumor in control group was greater than Evo group?1149.31±158.44mm³ VS324.21±22.36mm³?,the difference was statistically significant?P<0.01?.HE staining showed that the nucleus was atypia and account for a large proportion of the whole cell in control group,but Evo can significantly accelerate the nuclear pyknosis of cells in the tumor tissue and promote cell apoptosis.4.In vitro experiments were consistent with in vivo experiments.The ratio of LC3II/I was upregulated and the expression of p62 was decreased in HCT-116 cells and AMPK/mTOR signaling pathway in tumor cells treated with Evo were up-regulated and intracellular autophagy was activated.Conclusions1.Evo has an inhibitory effect on the proliferation of human colon cancer HCT-116 cells,and can inhibit the proliferation by activating autophagy when apoptosis is inhibited.2.In vivo and in vitro experiments showed that Evo could activate autophagy and inhibit the proliferation of colon cancer hct-116 cells by activating AMPK/mTOR signaling pathway.