Effect Of 5-fluorouracil And Aspirin Co-loaded Nanoparticles On Hepatocellular Carcinoma And Its Mechanism

ObjectiveHepatocellular carcinoma(HCC)is one of the most malignant tumors in the world.It ranks fifth in the global incidence of cancer.And it has the characteristics of strong invasion,easy metastasis and multiple lesions.Chemotherapy of liver cancer patients is easy to develop drug resistance,which is sensitive to radiotherapy.Therefore,it is the most important that we take safe and effective measures to inhibit the development of liver cancer,metastasis and anti-relapse.Clinical.Data showed that the expression of COX-2 protein in hepatocellular carcinoma was much higher than that in normal liver tissues.The patients with high expression of COX-2 had poorer prognosis,what suggest COX-2 may be a potential target protein for hepatocellular carcinoma.Aspirin is a kind of a non-steroidal anti-inflammatory drug.And it also is an cyclooxygenase inhibitor.It was used in antipyretic analgesics.Recently,studies shown that Aspirin has a role in anti-tumor.A combination of chemotherapy drugs can improve anti-tumor effect.It provides a new choice for cancer diagnosis and treatment.Therefore,in order to improve 5-fluorouracil antitumor efficacy and reduce side effects,this paper use hepatocellular carcinoma Hep G2,SMMC-7721,QGY cell line as the research object and prepare CS-based nanocarriers by co-delivery of 5-FU and Aspirin.Analyzed and studied on the features and their efficiency of 5-fluorouracil and Aspirin nanoparticles on proliferation,apoptosis,invasion and metastasis in hepatocellular carcinoma,and preliminarily explored the related mechanism,which will provide a new strategy for the treatment and prevention of liver cancer.MethodsFirst,HPLC was determined the content of 5-FU and Aspirin,and investigated its specificity,precision and recovery.Then,5-FACN was prepared by ion-gel method.This paper observed a series of physical and chemical properties,including appearance,particle size,zeta potential and entrapment efficiency.In addition,it also investigated the drug release of nanoparticles in vitro.The subcellular localization of FITC-loaded chitosan nanoparticles was detected by laser scanning confocal microscopy.Subsequently,ROS production measurement was examined the effect of blank NPs and reactive oxygen species inhibitor on the level of reactive oxygen species(ROS).Western blot analysis was detected the effect of each group on the invasion and metastasis related protein.And cell invasion and migration test was detected the effect of each group on the invasion and metastasis of QGY cell line.Then this paper investigated the antitumor effect of nanoparticles in vitro.MTT assay was used to detect the inhibitory effects of CN,5-FU and 5-FCN on the proliferation of hepatoma cells.And it selected almost the concentration of Aspirin as the combination concentration.The MTT assay and membrane protein V-FITC/PI staining kits what was used to detect the inhibitory effect of 5-FU,5-FCN,5-FCN+Aspirin and 5-FACN on the proliferation of hepatoma cells.PI staining kits was used to detect the effect of each group on the cell cycle.The expression of COX-2,NF-?B and apoptosis related protein Caspase-3,Bax were detected by Western blot.ELISA method was used to detect the content of PGE2 in the supernatant of hepatocellular carcinoma cells.Finally,we investigated the effect of co-loaded nanoparticles on invasion ability of SMMC-7721 and Hep G2 cells.ResultsThe specificity,linearity and precision of 5-FU and Aspirin were determined by HPLC assay,which accordance with the requirements of vitro assay.So it can be used to measure 5-FU and Aspirin content in vitro.CN,5-FCN and 5-FACN are uniform in size and structurally intact.They have a regular spherical or spheroid shape,good dispersibility and stability,and meet the requirements of size.Compared with CN,the loaded nanoparticles have no obvious change.It has a certain degree of sustained release in vitro,who has good dispersion and stability.The release behavior of 5-FCN,ACN and 5-FACN were not significantly different.It can be assumed that the release of 5-Fluorouracil and Aspirin in the loaded nanoparticles are independent of each other and do not interfere with each other.We observe the subcellular distribution of the nanoparticles into the cells by confocal laser scanning microscopy.It was found that they mainly gathered in the cytoplasm around the nucleus.CN was taken up by endocytosis and then entered the lysosomes.Cell invasion and migration experiments showed that NPs promoted the invasion and metastasis of cancer cells in a dose-dependent manner.When NPs and NAC were used in combination,the number of cells crossing the basement membrane of transwell cells who was significantly reduced compared with NPs group.These results showed that NPs can promote the invasion and metastasis of cancer cells,which may be related to NPs-mediated ROS levels.MTT and flow cytometry showed that drug-loaded nanoparticles could induce apoptosis significantly,especially 5-FACN.To a certain extent,co-loading nanoparticles blocked cells in the G1 / G0 phase.Western blot analysis results showed that the protein expression of Caspase-3 and Bax in hepatocellular carcinoma SMMC-7721 cells were significantly increased after 5-FACN treatment,indicated that Aspirin can synergistically promote the anti-tumor effect of 5-FU.After 5-FCN+Aspirin,5-FACN treatment,it was found that they could inhibit the invasion ability of SMMC-7721 and Hep G2 cells,and 5-FACN inhibited strongest.Conclusions5-FACN can effectively load the drug.In vitro it can inhibit the proliferation of hepatocellular carcinoma cells and induce apoptosis.The mechanism may be through down-regulation of NF-?B,thereby inhibiting COX-2 expression,reducing PGE2 synthesis and activating mitochondrial apoptotic pathway.Co-loaded 5-FACN can inhibit the invasion of liver cancer cells.The reason may be the inhibition of invasion and metastasis by inducing apoptosis.