Salvia Chinensia Benth.induces Autophagy In Esophageal Cancer Via AMPK/ULK1 Signaling Pathway

According to the global cancer incidence,mortality and prevalence report,esophageal cancer is the top 10 common type and cause of cancer-related death worldwide.In 2020,an i ARC--Cancer Today survey showed that 47.42%of esophageal cancer in the world occurred in China.Hebei Province,specififically Cixian County and Shexian County located to the south of the Taihang Mountains,has particularly high mortality and morbidity of esophageal cancer when compared to the rest of China and the world.Data have shown that people over 60 will mainly be affected.Esophageal carcinoma(EC)is divided into esophageal adenocarcinoma(EAC)and esophageal squamous cell carcinoma(ESCC).In Asia,especially China,ESCC is the most common histological type,accounting for more than 90% of esophageal cancers.Smoking and drinking are the main factors leading to the high incidence of esophageal cancer.In Asia,frequent drinking of extremely hot drinks and eating pickled vegetables are also high-risk factors.Conventional treatment for esophageal cancer is not effective and the 5-year survival rate of patients is only about 20%.Therefore,there is an urgent need to find new treatments to improve patient survival rates.In the treatment of cancer,Chinese herbal medicines have obvious benefifits: they support patient immunity,alleviate adverse effects of radiotherapy and chemotherapy,prevent recurrence and metastasis,improve patient quality of life,and prolong the survival rate.Many Chinese herbal monomers and their compounds have been found to have anticancer activities and inhibit esophageal cancer.Salvia chinensia Benth.(Shijianchuan in Chinese,SJC)is the whole grass of purple ginseng of Salvia genus in the Libidaceae family.It first appeared in the ancient Chinese medicine book Compendium of Materia Medica with the following functions: promotes blood circulation,removes stasis,reduces the mass and disperses knots.The Dictionary of Chinese Herbal Medicine records that SJC treats “Yege”,which is a traditional Chinese medicine term,manifested as dysphagia,or pain in swallowing,hard swallowing,or vomiting after eating,including esophageal cancer in modern medicine.In clinical practice,we found that SJC has a signifificant effect on the treatment of esophageal cancer,so it is worth further study as a potential therapeutic drug.Part One Based on proteomic analysis to explore the effect and mechanism of Salvia chinensia Benth.on esophageal cancer cellsObjective: To observe the effect of SJC on esophageal cancer cells,screen differential proteins with high throughput proteomics,and clarify the mechanism of SJC’s anti-esophageal cancer effect。Methods:1.The main chemical components in SJC were identified by Q-Orbitrap high-resolution LC/MS method.2.CCK-8 was used to detect the effect of SJC at different concentrations(0 μ g/m L,100 μ g/m L,200 μ g/m L,400 μ g/m L,800 μ g/m L,1600 μ g/m L,3200 μ g/m L)on esophageal cancer KYSE-150 cells and TE-1 cells,observe the sensitivity of the two cells to drugs,and calculate the IC50 value.3.Plate cloning method was used to detect the clustering ability of the two cells under SJC treatment with different concentrations(0 μ g/m L,62.5 μg/m L,125 μ g/m L,250 μ g/m L,500 μ g/m L).4.The method of tandem mass labeling and two-dimensional liquid chromatography-mass spectrometry(LC-MS)was used for proteomic and bioinformatics analysis using TMT labeling kit.Results:1.The identification results show that there are 28 components in SJC that are related to tumor,and five of them may be the components that SJC plays an anti-tumor role;2.SJC showed time-dependent and dose-dependent inhibition on esophageal cancer KYSE-150 cells and TE-1 cells.The IC50 values of KYSE-150 cells at 24 h,48h and 72 h were 538.5 μ g/m L,512.5 μ g/m L and371.4 μ g/m L,respectively.The IC50 values of TE-1 cells at 24 h,48h and 72 h were 1747 μ g/m L,1368 μ g/m L and 649.1 μ g/m L respectively.The inhibition effect of KYSE-150 cells is stronger than that of TE-1 cells,and the drug concentration reaching the half inhibition rate is lower;3.From the perspective of long-term culture,SJC effectively inhibited the cloning ability of esophageal cancer cells in a dose-dependent manner,so KYSE-150 cells were more sensitive to SJC,and KYSE-150 cells were selected for subsequent experiments;4.Proteomics and bioinformatics analysis showed that SJC could induce autophagy of esophageal cancer KYSE-150 cells and activate AMPK signaling pathway.Conclusions:1.SJC can inhibit the growth of esophageal cancer cells with different degrees of differentiation.2.SJC can regulate 1013 proteins of esophageal cancer KYSE-150 cells to produce statistically significant changes.3.The results of proteomics showed that SJC’s effect on esophageal cancer cells focused on autophagy,mitochondrial autophagy,lysosome,endocytosis,phagosomes and other cell pathways related to phagocytosis.4.SJC may promote the occurrence of autophagy through AMPK-ULK1 pathway,thus achieving the anti-tumor effect.。Part Two Salvia chinensia Benth.promotes autophagy of esophageal cancer cells through AMPK/ULK1 pathwayObjective: The proteomics results were verified by exploring the autophagy induction of SJC in vitro via the AMPK / ULK 1 pathway in esophageal cancer cells.Methods:1.Immunoblotting was used to observe the expression of AMPK-ULK1 signal pathway protein and autophagy classic protein LC3,P62.2.Laser confocal observation of fluorescent LC3 protein was used to reverse verify the existence of autophagy,and then AMPK inhibitor was used to block the upstream protein AMPK to observe the change of fluorescent LC3 and determine the mechanism of inducing autophagy.3.The autophagic body was captured by transmission electron microscope.Results:1.After SJC treatment,the expression of AMPK and autophagy promoter protein ULK1 increased,and the expression of P-AMPK and P-ULK1 also increased significantly.Compared with the control group,the transformation of LC3 in SJC treatment group increased significantly,and the expression level of P62 decreased;2.Compared with rapamycin(Rapa)group,500 μ G/ml SJC group and250 μ G/ml SJC group has autophagy promoting effect on KYSE-150 cells,500 μ G/ml SJC group had stronger promoting effect;3.At 500 μ The autophagy inhibitor chloroquine(CQ)partially blocked the enzymatic hydrolysis of autophagic bodies during the autophagy induction of esophageal cancer cells by g/m L SJC;4.AMPK inhibitor(Compound C)can partially inhibit 500 μAutophagy induction of esophageal cancer cells by g/m L SJC;5.The autophagic bodies in the early and middle and late stages were captured by transmission electron microscopy.Conclusions:1.SJC can up-regulate the autophagic classic protein LC3,and make it accumulate under the condition of blocking its decomposition.2.SJC can up-regulate AMPK,ULK1 and their phosphorylated proteins.With the addition of AMPK inhibitors,the autophagy induction of SJC on esophageal cancer cells is weakened,indicating that SJC induces autophagy of esophageal cancer cells through AMPK-ULK1 pathway.3.After SJC treatment,autophagic bodies were formed in esophageal cancer cells.Part Three The inhibitory effect and mechanism of Salvia chinensia Benth.on subcutaneous esophageal carcinoma in nude miceObjective:To explore the autophagy induction effect of SJC on esophageal cancer cells through AMPK/ULK1 pathway through in vivo experiments,and verify the results of proteomics and in vitro experiments.Methods:1.Esophageal cancer KYSE-150 cells were subcutaneously injected to construct a subcutaneous tumor model of esophageal cancer in nude mice.2.Nude mice were randomly divided into three groups: control group,low concentration group and high concentration group.SJC solution of different concentrations was given by gavage,and the tumor volume was measured every three days.Nude mice were killed for 21 days to weigh the tumor weight,frozen and fixed tissues for subsequent experiments.3.The autophagic body was captured by transmission electron microscope.4.The pathological changes of transplanted tumor in nude mice were observed by HE staining.5.The expression of autophagy related proteins ATG5,ATG7 and P62 was observed by immunohistochemistry.6.The expression of AMPK-ULK1 signal pathway and autophagic proteins LC3 and P62 were observed by immunoblotting.Results:1.Compared with the control group,the growth of transplanted tumor in the SJC low concentration group and the SJC high concentration group were inhibited to different degrees,and the inhibition effect of the SJC high concentration group was more obvious;2.The autophagic bodies and autophagic lysosomes were captured in the tumor tissues with different degrees of damage by transmission electron microscopy.3.After SJC treatment,the tumor tissue has different degrees of pathological changes.4.Immunohistochemical and Western blot tests showed that after SJC treatment,the expression of AMPK,P-AMPK,ULK1,P-ULK1,LC3,ATG5,ATG7 increased,and the expression of P62 decreased.Conclusions:1.SJC can inhibit the growth of transplanted tumor in nude mice after treatment.2.Autophagy bodies were formed in the transplanted tumor of nude mice after SJC treatment.3.SJC induces autophagy of transplanted tumor in nude mice,thereby inhibiting the growth of transplanted tumor.4.SJC induces autophagy in nude mice transplanted tumor through AMPK-ULK1 signal pathway.