The Role Of Autophagy In High Glucose Induced Proliferation Of Prostate Cancer Cells

Objective:To evaluate the role of autophagy in high glucose induced proliferation of prostate cancer cells by testing proliferation or apoptosis of prostate cancer cells and autophagy levels in high glucose condition. Then 3MA stimulate cell to observe change of cells. Method:1.The human prostate cancer cell lines LNCap were cultured in stable condition.Proliferation of LNCap was tested by CCK8 in different high glucose levels(normal level 5mmol/L,middle level 10mmol/L,high level 30mmol/L)and different time(24h,48 h,72h).Apoptosis of LNCap was tested by DAPI. Western blot measure the Bcl-2 proteins of LNCap.2.MDC、AO method test autophagy of LNCap in different high glucose(normal level 5mmol/L, middle level 10mmol/L, high level 30mmol/L) and different time(24h,48 h,72h).Western blot measure the LC3-Ⅱ/Ⅰ proteins of LNCap.Autophagy of LNCap was measured in different high glucose and different time by GFP-LC3 plasmid transfection.3. Choosing the 72 h and high glucose 30mmol/L condition to culture LNCap was considered as experimental subject.CCK8 test proliferation of cells and DAPI test apoptosis of cells when LNCap was cultured in high glucose condition stimulated by 3MA.Then to research the probable mechanism that blocking autophagy of prostate cancer in high glucose cultured condition affect proliferation and apoptosis of cells,we test ROS levels by flow cytometry and mitochondria potential by fluorescence microscopy. Results:1.CCK8 result show that proliferation of LNCap increase in high glucose condition; DAPI result show that apoptosis of LNCap keep low level in high glucose condition; Western blot result show that Bcl-2 protein is increasing with increased glucose levels and cultured time.Bcl-2 protein was expressed more in 72 h and high glucose condition.2. MDC、AO result show that autophagy of LNCap outstanding increase in high glucose condition. Western blot result show that autophagy protein LC3-II/I outstanding increase in high glucose condition. GFP-LC3 plasmid transfection experiment also prove those results.3. CCK8 result show that proliferation of LNCap was reduced in high glucose condition when 3MA of blocking autophagy agent stimulate LNCap. DAPI result show that apoptosis of LNCap increased in high glucose condition when 3MA of blocking autophagy agent stimulate LNCap. Flow cytometry result show that ROS of LNCap was increased in high glucose condition. Mitochondria potential test result show that 3MA can reduce LNCap mitochondria potential in high glucose cultured condition. Conclusion:High glucose can promote LNCap development and stimulate autophagy of LNCap increasing. The probable mechanism is that mitochondrial metabolism of cancer cell in high glucose make more destructive ROS, induce protective autophagy of cancer cell increasing. 3MA, blocking autophagy agent can destruct autophagy protective role, then leading to apoptosis of LNCap increasing in high glucose condition.