Effect Of Nitrite On Stem Cell-like Phenotype Characteristics Of Human Hepatocellular Carcinoma SMMC-7721 Cells

Background In most people's understanding,nitrite is the leading cause of cancer and has strong tumorigenetic.However many foreign studies had shown that nitrite has a good protective effect on cardiovascular disease.In recent years,with the development of tumor research deeply,there are a lot of evidence of primary or metastatic tumor tissues or cells existing in varying amounts similar to normal stem cells cancer stem cells(cancer stem cells,CSCs).However,our understanding of their regulation is limited.Rapid reversible changes of CSC-like cells within tumors may result from the effect of biological mediators found in the tumor microenvironment.,and even change the degree of malignancy of tumor cells or multiple drug resistance.In addition,the nitrite exists in theses biological mediators.Based on this controversy,it is particularly important to study the effect of nitrite on the regulation of tumor cells“stemness”.Aim This paper aimed to explore how nitrite,a key cellular modulator whose level is elevated in many tumors,affects CSC-like phenotypes of human hepatoma cells SMMC-7721 cells.Methods The SMMC-7721 cell line was cultured under serum-free conditions to produce floating spheres.The distribution of cell cycle and percentage of side population(sp)cells was analyzed by flow cytometry,the capability of cells self-renew were detected by colony-forming capabilities and spheroid-formation assay,the expression of stemness protein such as CD133,CD90 and Ep CAM were determined by flow cytometry and western blot,cell invasion was analyzed by transwell assay,and the viability of SMMC-7721 parental cells and spheroids cancer cells was determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide(MTT)assay.Results Our results showed that the SMMC-7721 spheroid cells were enriched with CSCs properties,indicated by the ability to self-renew,increased expression of CSCs markers,and increased resistance to chemotherapeutic drugs.Additionally,SMMC-7721 parental cells and spheroids cancer cells were treated with 16mg·L-1 sodium nitrite for 6 days,compared with control cells,an increased accumulation of G0/G1 phase cells was observable in treatment cells.Indeed,our data demonstrated that parent cells and spheres cells which were treated with sodium nitrite for different time,the effect of sodium nitrite on improve the number of SP,the cells' ability to chemoresistance and invasion,clone-forming efficiencies and the spheres forming ability were significantly higher than that of control cells.Exposure of sodium nitrite regulated CSC-like phenotype,indicated by increased expression of known CSC markers,CD133,CD90 and Ep CAM in the exposed parental cells,as well as in dormant spheroids cancer cells.Compared with the parent cells,the above effects of nitrite on the spheres cells were significantly enhanced.These results showed that nitrite could increase the invasiveness of SMMC-7721 cells through stemness features upregulation.Conclusions Low dose of nitrite can increase the stem cell phenotype of human hepatocellular carcinoma SMMC-7721 cells,which can enhance of malignancy and drug resistance,promote the migration and invasion.