| ObjectiveEsophageal carcinoma is the sixth most common cause of cancer-related deaths in the world.In developing countries,the major histological type of esophageal carcinoma is ESCC.In general,in most ESCC cases,the disease is at an advanced stage at the time of its diagnosis.Even in the early stage of ESCC,20% of patients experience a recurrence after curative esophagectomy.Curative surgery,chemotherapy and radiotherapy have limited effects on ESCC due to various factors,including tolerance,the general condition of the patient,tumor stage and treatment cost.To date,despite the wide variety of ESCC treatment options that are available,the poor prognosis of ESCC remains a challenge for clinical medicine.Therefore,developing a more effective treatment of ESCC would be highly desirableWith the advancement of science,many scholars have proposed the theory of cancer stem cell(CSC).The research on stem cell level has received more and more attention.As early as 1996,Margaret A.Goodell lived with bone marrow cells using fluorescent dye Hoechst 33342.After flow analysis,a group of double-negative cells outside the main group are obtained,which are called side population(SP)cells.This special cell group is highly tumorigenic,self-renewing,and more.The potential for differentiation and resistance is very similar to CSC.Therefore,SP sorting has become one of the identification methods of cancer stem cells,which lays a foundation for further study of the biological functions and molecular mechanisms of cancer stem cells.Metformin(Metf)has been widely used in the treatment of type 2 diabetes over the past 50 years and has been shown to not lower blood sugar in normal subjects.In recent years,the anti-tumor effect of Metf has been paid more and more attention.At present,the effect and mechanism of Metf on esophageal cancer CSCs are still unclear.Further research is needed.This study aims to study the dryness of metformin on esophageal squamous cell carcinoma SP cells.Role and mechanism.Methods1.Detection of SP ratio of esophageal squamous cell carcinoma cell lines(KYSE30,KYSE150,KYSE180,KYSE410,KYSE450,KYSE510,S1,TE1,Ec109)by flow cytometry;2.Comprehensive cell culture and SP ratio,KYSE150 was selected for cell sorting;3.CCK8 method,plate cloning and globization assay to detect the effect of metformin on the proliferation of SP cells in vitro;4.The effect of metformin on the expression of SP-related gene protein,the effect of metformin on SP cell apoptosis and cell cycle-associated protein,and the effect of metformin on PI3K/Akt and Stat3 signal transduction pathways in SP cells were detected by Western blot.5.SP and NSP cells treated with different concentrations of metformin were inoculated subcutaneously into the bilateral axillary fossa of nude mice to establish a subcutaneous xenograft model.After 30 days,the tumor formation and proliferation of different groups were compared.6.The experimental data were analyzed by SPSS19.0 statistical software.The Student t test and other test methods were used to make the map with GraphPad Prism 5.0 software.P<0.05 was considered statistically significant.Results1.In this experiment,the proportion of SP in 9 kinds of esophageal squamous cell carcinoma cells was detected.The SP ratio was about 0.2%~2%,and the SP ratios of different cell lines were different.2.After the KYSE150 cells were treated with different concentrations of metformin for 24 hours,the proportion of SP in the control group was(1.54±0.45)%,and the ratio of SP after 5mmol/L metformin for 24 h was(1.02±0.39)%,and 10mmol/L metformin 24 After the hour,the SP ratio decreased to(0.53±0.32)%,and the difference was statistically significant(P<0.05).After SP cells were sorted for different concentrations of metformin for 24 or 48 hours,cell proliferation was significantly inhibited,and the degree of cell proliferation inhibition was significantly positively correlated with metformin concentration and administration time.4.Plate colony formation: The average number of clones of 0 mmol/L metformin SP cells was(122.2±10.31),and the average clones of NSP cells were(51.4±4.58),but no clones were found in the medium supplemented with 5mmol/L metformin.The difference was statistically significant(P<0.01).5.Tumor Sphere Formation: The number of 0mmol/L metformin spheres in SP cells was(34.00±3.06),and the number of 5mmol/L metformin spheres was(12.67±2.03).The number of 0mmol/L metformin spheres in NSP cells was(13.00±1.53),and the number of 5mmol/L metformin spheres was(4.33±0.88).The difference was statistically significant(P<0.01).6.After the sorted SP cells were treated with different concentrations of metformin,the total protein was extracted,and the expression of stem cell related genes BMI1,SOX2 and OCT4 in the cells were found to be reduced to some extent.7.After treatment for 24 hours with different concentrations of metformin,the expression level of anti-apoptotic protein Bcl-2 was significantly lower than that of the control group,while the expression level of proapoptotic protein Bax did not change significantly,leading to promotion./ The proportion of anti-apoptotic proteins is increased.8.After treatment of the sorted SP cells with different concentrations of metformin for 24 hours,the expression of p-Stat3 and p-Akt was significantly inhibited,while the expression of Akt in the cells did not change significantly9.Xenograft mouse model:SP group had more tumors and larger volume than SP+Met,and the difference was statistically significant(P<0.01).Compared with NSP+Met,NSP group had more tumors and larger volume,and the difference was statistically significant(P<0.01).Conclusion1.Metformin can reduce the SP ratio of KYSE150 cell line,inhibit the proliferation of sorted SP cells,plate colony formation,spheroidization experiments,etc.,and can reduce the expression of stem cell-related genes such as BMI1,SOX2,OCT4,and with metformin concentration.The increase is more obvious,so it can be seen that metformin can reduce the dryness of esophageal cancer cells to some extent.This part of the experimental results confirmed that metformin may cause apoptosis and cell cycle arrest of SP cells by down-regulating the expression of Bcl-2 and cyclin D1.Metformin may also inhibit the proliferation of esophageal cancer SP cells through PI3K/Akt and Stat3 signaling pathways.Metformin inhibits SP cells from forming tumors。2.Metformin can kill cancer stem cells and reduce the proportion of cancer stem cells to a certain extent,which may provide a new idea for the clinical treatment of esophageal cancer,and provide a more effective treatment for patients with esophageal cancer. |
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