Study On The Effect And Mechanism Of Nano-TiO₂ Induced Endoplasmic Reticulum Stress On Apoptosis Of Tiver Cancer Cells

Research backgroundHepatocellular carcinoma,the main histological subtype of liver cancer,accounting for more than 90%of primary liver cancers,is the third most common cause of cancer-related mortality worldwide.The main treatment methods for liver cancer are liver transplantation,surgical resection and radio chemotherapy.However,the heterogeneity and drug resistance of liver cancer cells are the main barriers to the treatment of liver cancer.The recent research evidence suggests that the combination of nanoparticles with drugs or gene carriers can enhance the drug susceptibility of cancer cells.Nanometer Titania(Nano-TiO2)has aroused eager attention of researchers in the field of biomedicine because of its excellent chemical stability,biocompatibility,and photocatalysis.Nano-TiO2 affects cell function and induces the apoptosis of malignant tumor cells by promoting the production of reactive oxygen species(ROS).Nano-TiO2 can cause the oxidative stress in cells or tissues and preferentially damage cancer cells.However,there is still no in-depth research on the therapeutic effect of nano-TiO2 on liver cancer and its underlying mechanism.ROS-induced endoplasmic reticulum stress can trigger apoptosis through inducing the endoplasmic reticulum receptor protein such as protein endoplasmic reticulum kinase(PERK),transcription factor 6(ATF6),eukaryocyte initiation factor(eIF-2?)and X-box binding protein 1 and its downstream signaling pathways.It is reported that nano-TiO2 is involved in endoplasmic reticulum stress-mediated apoptosis of cancer cells.This study explored the toxic effect of nano-TiO2 on liver cancer cells and its mechanism to reveal its potential application in the treatment of patients with liver cancer.Objective(1)To define that the effect of nano-TiO2 on the growth of liver cancer cells and normal liver cells.(2)To define that the effect of nano-TiO2 on apoptosis of liver cancer cells and normal liver cells.(3)To explore the mechanism of endoplasmic reticulum stress signaling pathway in the induction of apoptosis of liver cancer cells by nano-TiO2.Method(1)Treatment of an experimental 6-well plate with a nano-TiO2 preparation solution at a concentration of 50ug/ml:?Weigh 5.0 mg of nano-TiO2 powder and sufficiently dissolve it in 100 ml of 75%ethanol solution;?Add 400?l of ethanol,100?l of nano-TiO2preparation solution+300?l of ethanol,200?l of nano-TiO2 preparation solution+200?l of alcohol,300?l of nano-TiO2 preparation solution+100?l of alcohol,and 400?l of nano-Ti02 preparation solution to each 6-well plate respectively;?Spread the suspension in the experimental well plate evenly on the bottom surface,and then place in an electric thermostatic drying oven at 60?;?After alcohol evaporates completely,remove the experimental well plate and place it in a clean bench,and irradiate with an ultraviolet lamp 2 h for standby.(2)Add a fixed number of human normal liver cell line LO2 cells and human liver cancer cell line HepG2 cells to 6-well plates treated with different concentrations of nano-TiO2 respectively,collect and treat the cells after 48 hours of culture,detect the cell growth,and observe the effect of nano-TiO2 on the growth of normal liver cells and liver cancer cells by cell counting method.(3)Add a fixed number of human normal liver cell line LO2 cells and human liver cancer cell line HepG2 cells to 6-well plates treated with different concentrations of nano-TiO2 respectively,collect and treat the cells after 48 hours of culture,detect the cell cycle distribution changes,apoptosis and intracellular ROS level by flow cytometry,and observe the effect of nano-TiO2 on the apoptosis of LO2 cells and HepG2 cells.(4)Add a fixed number of human normal liver cell line LO2 cells and human liver cancer cell line HepG2 and Bel-7405 cells to 6-well plates treated with different concentrations of nano-TiO2 or/and 4-PBA respectively,collect and treat the cells after 48 hours of culture,and extract the protein in the cells,analyze the expression level of ATF6,PERK and Bax proteins in the cells by Western Blot method to determine the process of nano-TiO2 inducing the apoptosis of liver cancer cells through endoplasmic reticulum stress.(5)Inject a fixed number of HepG2 cells into male BALB/c nude mice subcutaneously to establish a subcutaneous tumor model for in vivo animal experiments.According to the injection,HepG2 cells were divided into two groups:the control group treated with PBS and the experimental group treated with nano-TiO2.The diet,activity and weight of nude mice were observed every day.PBS and nano-TiO2 were administered every 6 days.After 7 weeks,the nude mice were euthanized.The effects of PBS and nano-TiO2 on subcutaneous tumorigenesis were observed and compared.Western blot and immunohistochemistry were used to compare the effects of PBS treatment and nano-TiO2 treatment on endoplasmic reticulum stress-related proteins in HepG2 cell metastasis.Result(1)Cell count analysis:Nano-TiO2 inhibited the growth of liver cancer cells,had no obvious effect on the growth of normal liver cells,and was concentration dependent.The difference was statistically significant(P<0.05).(2)Flow cytometry analysis:Nano-TiO2 increased the apoptosis of hepatocellular carcinoma cells and ROS levels,blocked the cell cycle in G1 phase,but had no significant effect on the apoptosis,ROS level,and cell cycle distribution of normal liver cells.The difference was statistically significant(P<0.05).Nano-TiO2 mediated the apoptosis in a dose-dependent manner.(3)Western Blot analysis:Nano-TiO2 had no significant effect on the expression of PERK,ATF6 and Bax in normal liver cells.However,western Blot analysis results showed that nano-TiO2 also increased the expression levels of PERK,ATF6 and Bax in liver cancer cells in a dose-dependent manner,and reversed by 4-PB A.(4)Animal experiments:In vivo administration of nano-TiO2 can reduce tumor volume and increase expression levels of PERK,ATF6 and Bax in tumor tissues.Conclusion(1)Nano-TiO2 can inhibit the growth of liver cells and arrest the cell cycle in G1 phase.And nano-TiO2 can produce ROS in liver cells and promote the apoptosis of liver cancer cells.(2)Nano-TiO2 can inhibit the growth rate of human liver cancer cell line in animal model,and the protein content of PERK,ATF6 and Bax protein increased significantly.(3)Nano-TiO2 can increase the protein content of PERK,ATF6 and Bax in a concentration dependent manner,and induces the apoptosis of liver cancer cells through endoplasmic reticulum stress.