| Objective To investigate the effects of lipoxygenase inhibitor,on proliferation and apoptosis in hepatocellular carcinoma cell line HepG2 and its apoptosis related genes in vitro,which we can elucidate the antineoplastic mechanisms of lipoxygenase inhibitors for the drug experiment and clinical application.Widening the research field of hepatocellular carcinoma in the pathogenesis and pharmacotherapeutics.Methods①A human hepatocellular carcinoma cell line,HepG2,was cultured in vitro and nordihydroguaiaretic acid(NDGA) was divided into 25,50,100,200μmol/L at concentration in the different times.Cells in logarithmic growth phase were selected and examined.②The proliferation inhibition was analysed by alive cell count,MTT assay.③Cells apoptosis was observed with inverted microscope,Hoechst33258 labeling and transmission electron microscope.④Cell cycle analysis,apoptosis rate were evaluated by Annexin V/PI and flow cytometry(FCW)method.⑤The mRNA expression of apoptosis related genes telomerse,bcl-2 and bax were examined by RT-PCR.Results①NDGA can inhibit HepG2 cells proliferation viability within a certain range (25～200μmol/L) of treating time and dose,in which the inhibition increased from 5.23%to 61.20%.The higher the concentration of NDGA was,the stronger the cytotoxic effect reached,which suggested obvious dose-dependent manner of NDGA;The inhibitory effects were augmented with the prolongation of culture(24h,48h,72h),which had time-dependence.The r values of dose-effect curves for NDGA on HepG2 cell line were 0.965,0.902 and 0.981 respectively.The IC50 values were 128.37μmol/L(72h).High concentration NDGA can induce the apoptosis of HepG2.②Under the Hoechst33258 labeling,the cell morphology obviously changed treated with different concentrations of NDGA for 48h.Some cells became much more round and brighter,shrinking with the membrane crimpled like burrs.The cytoplasm contained highly concentrated chromosomes which attached to the nuclear membrane and came into the shape of clump,crescent or fragment.Many cells fell off the wall of medium and were suspensive in the culture medium.③Under the scanning electron microscope,HepG2 Cell showed typical apoptotic morphological changes after treatment of NDGA,characterized by cellular roundness,volume shrinkage and membranous crinkle.The changes of microvillus and filopodium were very distinct.The filopodia were broken,and the microvilla were curled and shortened.Some cells nuclear condensated and apoptosis bodies were formed.④With the increase in the concentrations of NDGA,the HepG2 cells apoposis increased gradually After 48h treatment(200μmol/L),the early apoptosis reached 41.6±2.3.NDGA can induced cell cycle arrest at the S phase in HepG2 cell lines,along with the decrease in the population of G0/G1 phase cells.⑤The expression of bax mRNA was up-regulated and down-regulated of 5-LOX mRNA,bcl-2 mRNA and hTERT mRNA in HepG2 cells by RT-PCR after NDGA treatment for 48h,all the expressions of mRNA with dose-related effect and were compared significantly(P＜0.001).Conclusion①NDGA can inhibited HepG2 cells proliferation in a time- and dose-dependent manner,and cells proliferation was blocked in the S phase.The mechanism may be implemented though inducing apoptosis and preventing proliferention.②Because NDGA has a dramatical effect on the morphology of HepG2 cells,especially on the filopodia and microvilla,we can conclude NDGA might prevent the adhesion and invasion of the ceils.③The expression of 5-LOX mRNA was strongly positive in HepG2 cells.One of the mechanisms of NDGA inhibition proliferation and inducing apoptosis is likely to connect with the down-regulation of 5-LOX mRNA.④The effect of NGDA on cells proliferation and induced the apoptosis which may be associated with the upregulating expression of bax mRNA and downregulating expression of hTERT mRNA,bcl-2 mRNA.Lipoxygenase might be a novel therapeutic target for the hepatocellular carcinoma.
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