Inhibitory Effect And Mechanisms Of Iturin On Hepatocellular Carcinoma

In recent years,the incidence and mortality of hepatocellular carcinoma(HCC)have remained high all over the world due to the lack of obvious initial symptoms,the difficulty of late treatment and the low prognostic survival rate.The development of anti-hepatocellular carcinoma drugs with the characteristics of multi-target,non-drug resistance and low toxicity and side effects has become an urgent problem to be solved.In this paper,we researched the iturin produced by Bacillus subtilis and its inhibitory effect on hepatocellular carcinoma and mechanism.The main research contents and results are as follows:(1)Anti-cancer activity analysis and molecular identification of lipopeptide.The inhibitory effects of different components of lipopeptides produced by Bacillus subtilis on many kinds of tumor cells were detected,and it was found that the main active component was iturin.Further analysis showed that the inhibitory effect of iturin on the growth of HepG2 cells was concentration-dependent.The IC50 value of iturin to HepG2 cells was 11.91 ?M.And it could completely inhibit the growth of HepG2 cells at 60 ?M.(2)The mechanism of inhibiting HepG2 cells by iturin in two-dimensional culture was analyzed.It was found that iturin could rapidly enter HepG2 cells and induce apoptosis,paraptosis and autophagy at the concentration of IC50.Among them,apoptosis was endogenous mitochondrial pathway,which showed as DNA damage,accumulation of intracellular reactive oxygen species,decreased mitochondrial membrane potential,enhancement of mitochondrial membrane permeability,upregulation of the expression of p53,bax and bad proteins,down-regulation of bcl-2protein expression,promotion of release of cytochrome C from mitochondria,activation of caspase.The phenomena of paraptosis were as follows: a large number of vacuoles appeared in HepG2 cells,which triggered endoplasmic reticulum stress response,resulting in endoplasmic reticulum dysfunction,endoplasmic reticulum swelling,a large amount of calcium ion outflow,and swelling of mitochondria.The characteristics of autophagy were: increased ROS level in cells,production of a large number of autophages,up-regulation of LC3 II expression,but up-regulation of p62,which showed that autophagy was triggered,but the progress was inhibited at last.(3)The mechanism of inhibiting HepG2 cells by iturin in three-dimensional culture was analyzed.The results showed that the growth of HepG2 in threedimensional culture was still inhibited by iturin in a concentration-dependent manner.The IC50 value was 55.26 M.The mechanism analysis showed that iturin could induce apoptosis of endogenous mitochondrial pathway as nuclear chromosome agglutination,ROS accumulation,cytochrome c release,etc.Meanwhile,it triggered autophagy,which was shown as autophagic bodies appeared,up-regulation of LC3 II and upregulation of p62.Unlike the results of two-dimensional culture mode,in the threedimensional culture mode iturin didn't induce paraptosis,nor did it block cell autophagy.(4)Safety evaluation of oral iturin and therapeutic effect analysis of hepatocellular carcinoma xenograft model.7 days acute toxicity evaluation results showed that oral administration of 5000 mg/kg(bw)of iturin didn't produce acute toxicity in mice;oral administration of 2000 mg/kg(bw)of iturin for 28 days did not cause obvious organ damage and abnormal blood indexes in mice,and could significantly reduce the abundance of intestinal Firmicutes,increase the abundance of Bacteroides,cause Bifidobacterium and Butyricimonas and other benefits.The intestinal microflora in mice showed a certain improvement.However,oral administration of 3 mg/kg(bw)could not effectively inhibit the increase of liver cancer volume and weight in the xenograft model of human hepatocellular carcinoma mice,and had no significant therapeutic effect.(5)Therapeutic effect of subcutaneous injection of iturin on hepatocellular carcinoma xenograft model.By injecting 3 mg/kg(bw),the growth of xenograft tumors in mice was significantly inhibited by iturin,and the inhibition rate was 58.55%.It was found that iturin could significantly reduce the expression of TGF-?1 in tumor tissues,thereby reducing the possibility of immune escape of tumor cells.At the same time,iturin could also induce apoptosis of endogenous mitochondrial pathway in tumor cells.The activities of alanine aminotransferase and glutamic oxalate aminotransferase and the content of creatinine were increased in serum of mice in the treatment group.Slight hyperemia of liver was observed in tissue sections,and no obvious kidney damage was observed.In this paper,we not only found the potential of iturin in inhibiting hepatocellular carcinoma in vitro and in vivo,but also revealed its pathways and mechanisms,and evaluated the safety and effectiveness of oral administration and injection of iturin.This study is innovative in revealing the apoptosis and autophagy of hepatocellular carcinoma cells induced by iturin.The results of this study can provide theoretical guidance and practical basis for iturin in the treatment of cancer.