Mechanism Of 6-ethoxyxosthetine In Inhibiting The Proliferation Of Esophageal Squamous Cell Carcinoma

Esophageal cancer is one of the most common malignant tumors,ranking sixth among all malignant tumors in worldwide.The number of patients and deaths of esophageal cancer in China accounts for about 50%of the world.According to the type of pathology,esophageal cancer can be divided into esophageal adenocarcinoma(EAC)and esophageal squamous cell carcinoma(ESCC)and undifferentiated cancer.In China,about 90%of esophageal cancer patients are ESCC.The current treatment options are roughly divided into:surgical resection,radiation therapy,and chemotherapy,but these methods often have disadvantages such as low cure rate and large side effects.Due to the lack of effective treatment methods,the five-year survival rate of patients with esophageal cancer is less than 20%.Therefore,it is very important to find natural compounds with high efficiency and low toxicity for the treatment of esophageal cancer.This topic screened a variety of natural compounds through cytotoxicity experiments and found that 6-Ethoxysanguinarine is cytotoxic to ESCC cells and has no significant toxic effect on normal esophageal epithelial cells.6-Ethoxysanguinarine is a natural compound that can be extracted from Boroide(Papaveraceae)and belongs to benzophenanthridine alkaloids.Untill now,there are no reports about the effect and mechanism of 6-Ethoxysanguinarine on ESCC.In this study,we first demonstrated that 6-Ethoxysanguinarine can inhibit the proliferation and anchorage-independent growth of ESCC cells through cell phenotypic experiments.In order to explore the mechanism of this inhibition,we conducted phosphorylation proteomics analysis and found that after treatment with 6-Ethoxysanguinarine,the Adherens junction,Ribosome biogenesis in eukaryotes,Pertussis,mTOR signaling pathway,RNA degradation,N-Glycan biosynthesis pathways were down-regulated,and the phosphorylation levels of MAPK3Y204 and MAPK1Y187 found in these pathways were frequently and Down-regulated,which were verified by Western blotting.In addition,Western blotting also verified that the phosphorylation levels of p-CRAFS338 and p-MEKT394 were reduced on the MEK/ERK pathway,the phosphorylation level of p-EGFRT693 haven’t changed,and The PI3K/AKT pathway which is related to EGFR protein is also downregulated.Through western blotting experiments and computer molecular simulation experiments,EGFR was found to be a potential target.In order to verify this conjecture,EGFR kinase activity was first tested.Compared with the control group,it was found that 6-Ethoxysanguinarine action can make EGFR kinase activity Reduced.Then the Pull-Down experiment and the surface plasmon resonance experiment were performed.and The results showed that 6-Ethoxysanguinarine can bind with EGFR.Furthermore,the EGFR-knockdown cell lines were established to observe the changes of cell number in the proliferation of ESCC.EGFR-knockdown cell lines were treated with 6-Ethoxysanguinarine,the results revealed that the EGFR-knockdown cell lines by treating with 6-Ethoxysanguinarine had a higher survival rate than wild-type ESCC cells,The inhibitory effect of 6-Ethoxysanguinarine on EGFR-knockdown cell lines were decreased,indicating that EGFR is the target of 6-Ethoxysanguinarine and may be a new type of EGFR inhibitor candidate,which can be used as a potential chemoprevention for esophageal cancer drug.More deeply,the study found that 6-Ethoxysanguinarine can inhibit tumor growth in SCID mice in a humanized esophageal squamous cell carcinoma xenograft(PDX)model in vivo.Based on in vivo experiments,immunohistochemical experiments were performed,and the results showed that the positive cell rates of p-CRAFS338 and p-PI3KT467 in the 6-Ethoxysanguinarine treatment group were significantly reduced.Materials and methods1.Cytotoxicity experiment:To study the toxic effects of 6-Ethoxysanguinarine on human esophageal squamous cell carcinoma cells KYSE150 and KYSE450 and normal esophageal epithelial cells SHEE.2.Cell proliferation experiment:To investigate whether 6-Ethoxysanguinarine can inhibit proliferation of human esophageal squamous cell carcinoma cells KYSE150 and KYSE450 and normal esophageal epithelial cells SHEE.3.Soft agar experiment:To investigate whether 6-Ethoxysanguinarine can inhibit the formation of soft agar clones of esophageal squamous carcinoma cells.4.Phosphorylation proteomics experiments:To find the changes of intracellular phosphorylation modification sites induced by 6-Ethoxysanguinarine treatment of KYSE150 cells,and analyze its mechanism of inhibiting the proliferation of human esophageal squamous cell carcinoma.5.Western blotting experiments:Validate p-CRAFS338,p-MEKT394,p-ERKT185/Y187,p-ERKT202/Y204,p-EGFRT693 and Whether the phosphorylation level of PI3KT467,p-AKTS473 has changed.6.Pull-down experiment:To study whether 6-Ethoxysanguinarine and EGFR in KYSE150 cell lysate can be linked each other.7.Surface plasmon resonance experiment(SPR):Detect the binding kinetics of 6-Ethoxysanguinarine and EGFR pure protein.8.Test for detecting EGFR kinase activity:To investigate whether the activity of EGFR kinase decreases after treating esophageal squamous cell carcinoma cells with 6-Ethoxysanguinarine.9.Knockdown experiment:knock down the EGFR gene in the cells,and test whether the survival rate of the knocked-down cells after adding 6-Ethoxysanguinarine is compared with the control to see if there is still an inhibitory effect.10.PDX model:To study the inhibitory effect of 6-Ethoxysanguinarine on human esophageal squamous cell carcinoma in SCID mice.Results1.Cytotoxicity test show that the concentration at 1 μM of 6-Ethoxysanguinarine can make the cell survival rate of KYSE150 and KYSE450 become 50%,and almost has no toxic effect on SHEE cells.2.The results of cell proliferation experiments show that when the concentration of 6-Ethoxysanguinarine is less than 1 μM,both KYSE150 and KYSE450 cells have a significant inhibitory effect on proliferation,but have no effect on the proliferation of SHEE cells.3.The results of soft agar clone formation experiments showed that when the concentration of 6-Ethoxysanguinarine was 1 μM,it significantly inhibited the anchor-independent growth of KYSE150 and KYSE450 cells.4.Phosphorylation proteomics results show that 6-Ethoxysanguinarine can significantly down-regulate Adherens junction,Ribosome biogenesis in eukaryotes,Pertussis,mTOR signaling pathway,RNA degradation,N-Glycan biosynthesis and other pathways MAPK3Y204 and MAPK1Y187 acidification levels in these pathways.5.Western blotting results showed that p-CRAFS338,p-MEKT394,p-ERKT185/Y187,p-ERKT202/Y204,p-PI3KT467,p-AKTS473 protein expression levels were significantly reduced.6.The results of the Pull-down experiment show that 6-Ethoxysanguinarine can bind to EGFR.7.The results of SPR experiments showed that the determination of the KD value of the affinity constant between 6-Ethoxysanguinarine and EGFR pure protein was 5.263E-4 mM,indicating that 6-Ethoxysanguinarine can effectively bind to EGFR,8.EGFR kinase activity test results showed that the EGFR kinase activity in the 6-Ethoxysanguinarine-treated group was significantly reduced compared to the control group.9.Knockdown results show that the survival rate of KYSE150 and KYSE450 cells that knock down the EGFR gene is higher than that of wild-type ESCC after adding 6-Ethoxysanguinarine,indicating that EGFR is 6-Ethoxysanguinarine The target of 6-Ethoxysanguinarine.10.The results of the PDX model showed that the tumor volume and weight of the 6-Ethoxysanguinarine administration group were significantly lower than those of the untreated group and were statistically significant.Conclusion6-Ethoxysanguistine can inhibit MEK/ERK and PI3K/AKT signaling pathways by binding EGFR,thereby exerting the effect of inhibiting ESCC cell proliferation.