Exdogenous HMGB1 Play A Important Role In The Proliferation And Metastasis Of Surviving Pancreatic Cancer Cells After Radiotherapy

OBJECTIVE To study the role of exdogenous HMGB1 in the metastasis of surviving pancreatic cancer cells.In this study,we need to find the molecular mechanism of exdogenous HMGB1 induced proliferation and metastasis of surviving pancreatic cancer cells.METHODS(1)ELISA assay:SW1990 and PANC-1 cells were seed into 24-well culture plates at the density of 1×105cells per well and at the capacity of 800?l per well.In this study,we need 10 culture plates.All SW1990 and PANC-1 cells which were ready treated with different radiation dosage(0Gy,4Gy,8Gy,10 Gy,12Gy).These cells were continued to culture in Rsbiotech for 24 h after radiotherapy.Then our team extract all supernatant of 5 groups and detect the content of HMGB1 in these supernatant.The data is statistically analyzed.(2)Collected images by EMS: SW1990 and PANC-1 cells were seed into 24-well culture plates at the density of 1×105 cells per well.We need 4 culture plates in this experiment.All SW1990 cells which were ready divided into groups of different radiation dosage(0Gy,10Gy),and all PANC-1 cells which were ready divided into groups of different radiation dosage(0Gy,4Gy).Then these cells were continued to culture in Rsbiotech for 24 h after radiotherapy.After exchange of the cell medium,we need to collect all images by electron microscope(EMS).(3)Wound healing assay:surviving SW1990 cells and surviving PANC-1 cells after radiotherapy were seed into 24-well culture plates at the density of 1×105cells per well.We need 6 culture plates in this experiment.When surviving SW1990 cells and surviving PANC-1 cells were all ready,we need to scratch.After washing 3 times by PBS,surviving SW1990 cells and surviving PANC-1 cells were random Ly divided into 3 groups which were control group,HMGB1 group and HMGB1+EP group.At the end,we need to collect all images by Optical Microscope(OM)at different time intervals(0h,12 h,24h).(4)CFSE assay: surviving SW1990 cells and surviving PANC-1 cells after radiotherapy were seed into petri dish(10cm diameter).In this study,we need 9 petridish for every cells.All surviving SW1990 cells and surviving PANC-1 cells which were ready divided into 3 groups which were including control group,HMGB1 group and HMGB1+EP group.Then these surviving cells were continued to culture in Rsbiotech for 24 h.And then analyze the different effect on the proliferation of surviving SW1990 cells and surviving PANC-1 cells by CFSE.(5)Western Blot analysis:surviving SW1990?PANC-1 cells after radiotherapy were seed into petri dish(10cm diameter).We need 4 petri dish in this experiment.When surviving SW1990 cells and surviving PANC-1 cells were all ready,All surviving SW1990 cells and surviving PANC-1 cells which were ready divided into 4 groups:control group of surviving SW1990 cells,HMGB1 group of SW1990 cells,control group of surviving Panc-1 cells and HMGB1 group of Panc-1 cells.Then surviving SW1990 cells and surviving PANC-1 cells were continued to culture in Rsbiotech for24 h.Then analyzed protein expression level of Ki67,Bcl-2,E-CA,N-CA,GSK-3?,p-GSK.(6)In vivo: SW1990 cells were seed into petri dish(10cm diameter).We need 9 petri dish in this study.When the density of SW1990 cells is close to 100%,we inject 200?l cell suspension under the skin of nude mice after the digestion and centrifugation of SW1990 cells.After build the subcutaneous mouse model successfully,9 nude mice need radiotherapy of 10 Gy per time,every two days.Then 9 nude mice were ready divided into 3 groups which were control group,HMGB1 group and HMGB1+EP group.Measured the volume of subcutaneous tumors every two days and analyzed the data by Graph Pad.RESULTS(1)The content of HMGB1 in supernatant by different radiation dosage(4Gy,8Gy,10 Gy,12Gy)were more higher vs control group.When radiation dosage is10 Gy,the content of HMGB1 in supernatant of SW1990 cells is the highest.When radiation dosage is 4Gy,the content of HMGB1 in supernatant of Panc-1 cells is the highest.(2)Normal SW1990 cells and PANC-1 cells morphology are fusiform cultured in the normal medium.However,the volume of morphology is more bigger than normal SW1990 and PANC-1 cells.And the growth velocity of surviving SW1990 cells and surviving PANC-1 cells are more faster than normal SW1990 cells and normal PANC-1 cells.(3)The migration distance and the migration area of the surviving SW1990 cells and surviving PANC-1 cells were obviously increased in HMGB1 group vs control group and HMGB1+EP group,detected by Wound healing assay.However,the migration distance and the migration area of the surviving SW1990 cells and surviving PANC-1cells were obviously shrink in HMGB1+EP group than control group by Wound healing assay.(4)The proliferation rate of the surviving SW1990 cells in three groups(control group,HMGB1 group and HMGB1+EP group)were(43.6±2.1)%,(62.5±2.1)%and(33.2±1.7)% by CFSE.And the proliferation rate of the surviving Panc-1 cells in three groups(control group,HMGB1 group and HMGB1+EP group)were(37.5 ±0.9)%,(49±1.4)% and(29.3±1.3)% by CFSE.The proliferation rate of HMGB1 group is more higher than other two groups.(5)Exdogenous HMGB1 can up-regular the expressions of N-CA,Ki67,Bcl-2 and p-GSK proteins and down-regular the expressions of E-CA,which had statistical significance compared with the control group.CONCLUSION Radiation can induce HMGB1 released from surviving pancreatic cancer cells after radiotherapy.Exdogenous HMGB1 play a important role in the proliferation and metastasis of surviving pancreatic cancer cells after radiotherapy.Exdogenous HMGB1 can up-regular the expressions of N-CA,Ki67,Bcl-2 and p-GSK proteins and down-regular the expressions of E-CA.Exdogenous HMGB1 is related with the metastasis mechanism and EMT of surviving pancreatic cancer cells.