Effect Of TTF1-NP On The Biological Behavior Of Hepatocelluar Cancinma Cells Through STAT3 Regulation

Background:Primary hepatocellular carcinoma is one of the most common malignant tumors,which is a serious threat to human health.The mortality rate of primary liver cancer has been increasing year by year,while a decreasing mortality trend is observed for malignant tumors across the world.China has a high liver cancer incidence rates,the annual mortality of primary liver cancer in China is about 4.6 million,which accounts for 1/2 of the liver cancer deaths in the world.Therefore,it is of great practical significance to search for molecular targets and therapeutic drugs for the treatment of liver cancer.Signal transducer and activator of transcription 3(STAT3)is closely related to the occurrence and development of many kinds of tumors,and detected in approximately 70%of malignant tumors.STAT3 is a transcription factor,known as an attractive target for cancer therapy.TTF1 nanoparticles(TTF1-NP)are a biodegradable and small molecules,which were prepared using an emulsion evaporation-solidification method from TTF1,the major anticancer bioactive constituent of Sorbaria sorbifolia.TTF1-NP can inhibit the proliferation of various tumor cells by regulating the apoptosis-assoicated proteins and affecting the tumor cell cycle.These in vitro studies provide a strong biochemical rationale for a further research and encourage us to investigate its anticancer mechanisms.Objective:In this study,we investigated the effect of TTF1-NP on the protein expression of STAT3 in human hepatocellular carcinoma cells,detected the effects of TTF1-NP on the angiogenesis,invasion,migration,and apoptosis of hepatocarcinoma cells,and explored the molecular mechanism of STAT3-dependent biological behavior regulated by TTF1-NP so as to provide the theoretical basis for the application of TTF1-NP for liver cancer.Methods:The inhibitory effect of TTF1-NP on the growth of human hepatoma cells and the protein expressions of STAT3 and p-STAT3 were detected in different human hepatoma cell lines and the nude mice model transplanted with HepG2 cells;the binding activity of STATS and its target DNA was detected by EMSA;the angiogenesis,invasion,migration,and apoptosis of HepG2 cells were detected by tube formation assy,HE and Hoechst staining,flow cytometry,transwell and cell scratch assays;the expressions of related functional proteins were detected by western blot;HepG2 cells with STAT3 knock-down or over-expression were established to explore the regulatory mechanism of TTF1-NP on the biological behavior of human hepatocellular carcinoma HepG2 cells.Results:1.The results showed that TTF1-NP inhibited the proliferation of human hepatocellular carcinoma HepG2,Hep3B,PLC/PRF/5 and SMMC-7721 cells in a dose-and time-dependent manner,with the IC50 values of 99.3,119.4,147.8 and 121.5}imol/L at 48 h,respectively,and HepG2 cells was the susceptible one among them.TTF1-NP also showed an inhibition effect on the growth of HepG2 transplanted tumor tissue in nude mice with the growth inhibition rates of(48.9±4.7)%,(52.9±3.5)%and(58.8±5.4)%,respectively at the doses of 5?mol/kg,10 ?mol/kg and 20 ?mol/kg.2.Immunochemical and western blot assay showed that STAT3 and p-STAT3 protein expressions were significantly reduced in response to TTF1-NP in HepG2 cells and nude mice in a dose-dependent manner;TTF1-NP significantly inhibited the binding of STAT3 and its target DNA in a dose-dependent manner.3.TTF1-NP markedly decreased HUVEC microtube formation,and inhibited the expression of VEGF,KDR and bFGF proteins in HepG2 cells;TTF1-NP also inhibited the migration and invasion abilities of HepG2 cells in a dose-dependent manner,as well as the expression of MMP2 and MMP9 proteins;TTF1-NP induced apoptosis,and promoted the expression of cleaved caspase3 while inhibited the expression of survivin.4.HepG2 cells with STAT3 knock-down and over-expression were constructed respectively.TTF1-NP showed no further inhibition effect on the biological behavior and the expression of related functional proteins in HepG2 cells with STAT3 knock-down;on the contrary,TTF1-NP showed a significant inhibitory effect in HepG2 cells with STAT3 over-expression.Conclusions:1.TTF1-NP inhibited the phosphorylation of STAT3,and decreased the DNA-binding capacity of STAT3.2.TTF1-NP suppressed prolifition,angiogenesis,invasion and migration,and induced apoptosis of HepG2 cells through STAT3 pathway.3.TTF1-NP could suppress angiogenesis,invasion and migration,and induce apoptosis of HepG2 cells through STAT3 pathway,the possible mechanisms may be associated with regulation of VEGF,KDR,bFGF,MMP2,MMP9,survivin and cleaved caspase3.