The Mechanism Of Chitooligosaccharides Inhibiting HepG2 Tumor Cells

Chitooligosaccharides(COS)are oligosaccharides(and their salts)formed by linking D-glucosamine(or a small amount of N-acetyl-D-glucosamine)through β-1,4-glycosidic bonds.Degree of polymerization(DP)is generally distributed between 2 and 10.COS have various bioactivities,among which the anti-tumor properity is highly concerned.However,due to the lack of technologyin preparaing COS with singular DP,the effect and mechanism of chitosan oligosaccharide inhibition of tumor cells is still unclear.This paper aimed to study the anti-proliferation effect of COS using singular COS(DP 2-5),observethe phenotype changes after being incubated with singular COS,and clarify the molecular mechanism of inhibiting HepG2 cell proliferation.Firstly,high performance liquid chromatography(HPLC)was used to analyse the consitituent of single COS with NH2P-40 4E column and ELSD detector.Gradient elution was uesd to perform the analysis.The results showed that each single COS have one main peak.The purity of each single COS was above 90%.Secondly,HepG2 cells were incubated with singular COS with different concentrations.MTT assay was performed to detect the inhibition rate.IC50 values were calculated to determine the inhibition effect of each singular COS.The results showed that cell proliferation rate decrease significantly with(GlcN)2,(GlcN)3at 4mg/mL,(GlcN)4at 2mg/mL,(GlcN)5at lmg/mL(p<0.05).The IC50 values of(GlcN)2-(GlcN)5 were 7.18、7.52、6.49、3.49mg/mL,respectively.The results indicated that(GlcN)5have the best anti-proliferation effect.The relative cell viability of human normal liver cell L02 when treated with 7mg/mL of chitopentaose was 72.06%.Thirdly,Annexin V-FITC/PI double staining with flow cytometry were used to detect the change of apoptosis rate after cells were being incubated with 3.5mM singular COS.The results showed that the apoptosis rate increase as the increase of DP.The apoptosis rate were 16.57、17.23、20.39、24.57%,respectively.Morphological change was observed after cells were treated with 3.5mM(GluN)5 using an inverted microscope.The results showed that cells become smaller,rounder.Shrinkage,a hallmark of apoptosis,as well as increasement of gaps between cells were also observed.Nuclear moiphological change was observed as well.After cells being incubated with(GlcN)5,nuclear condensation and fragmentation were observed.The apoptosis level increased as(GluN)5 concentration increased in a dose-dependent manner.JC-1 staining was used to study the mitochondrial membrane potential(MMP).Results showed that with the treatment of(GluN)5,MMP decreased in a dose-dependent manner.RT-qPCR and Western Blot were performed to detect the mRNA and protein levels.Results showed that Bak and Bax mRNA levels increased significantly.The protein levels of Bax,Cleaved Caspase-9,Cleaved Caspase-3,Cleaved PARP,and cytoplasmic Cytochrome C increased significantly,Bcl-2 decreased significantly.The ratio of Bax/Bcl-2 increased,indicating apoptosis.The results above indicated that(GlcN)5 could induce apoptosis via mitochondrial pathwayFinally,HepG2 was incubated with 3.5mM of(GlcN)5,and the autophagy levels were studied.Transmission electron microscopy and immunofluorescence results showed that(G1cN)5 could induce the accumulation of autophagosomes.Western Blot results showed an increase in p62 and ratio of LC3-II/LC3-I,but Beclinl had no significant change,indicating that(GlcN)5 might impair the autophagy flux and the degragation of autophagosomes.Chloroquine(CQ),an inhibitor of autophagic flux,was used to further study the status of autophagy flux.Results showed(GlcN)5 had similar effect with CQ.Both of them could increase the accumulation of autophagosomes,impair the fusion of autophagosomes and lysosomes,as well as increase the expression of p62 and the ratio of LC3-Ⅱ/LC3-I Furthermore,the cell viability and apoptosis levels were studied under the circumstance of impaired autophagy flux.A decrease in cell viability and an increase in apoptosis rate were found when autophagy flux was impaired by(GlcN)5 or CQ.Moreover,an increase in the relative expression level of Cleaved Caspase-9,Cleaved Caspase-3,and and Cleaved PARP was found.These results indicate that(GlcN)5 could inhibit the fusion of autophagosomes and lysosomes andprevent the protective autophagy in HepG2,then promote the apoptosis to inhibit thecell proliferation.To sum up,(GlcN)5 could inhibit HepG2 proliferationthrouth two molecular mechanisms:1.Inducing apoptosis through mitochondrial pathway;2.Impairing the fusion of autophagosomes and lysosomes and impairing the autophagy flux.