| Bovine lactoferricin(LFB)is an active peptide produced by the hydrolysis of bovine lactoferrin under acidic conditions.It has nonspecificability to combine with cancer cells carrying a large number of negative charges because of its 8 positive charges.Bovine lactoferricin has almost no toxicity to normal cells in a concentration range that exerts antitumor activity to many tumor cell lines.The purpose of this study is to verify whether LFB has a selective toxic effect on hepatocellular carcinoma and to study its mechanism of producing anti-tumor activity against Bel-7402 and Huh-7 cells in vitro,providing a reference for the anti-hepatocellular carcinoma activity of LFB.The toxic effects of synthetic LFB on liver cancer cells Huh-7,Bel-7402 and human normal hepatocytes QSG-7701,L02 were detected by CCK-8 method.The four types of cells were treated with LFB of 0,0.2mg / m L,0.4 mg / m L,0.8 mg / m L,1.6 mg / m L,and 3.2 mg / m L;cell plate cloning experiments were performed with untreated Huh-7 and Bel-7402 as blank control groups.The groups treated by LFB with concentrations of 0.6 mg / m L,0.8 mg / m L and 1.0 mg / m L were used as the experimental groups to measure the proliferation inhibition rate of Huh-7 and Bel-7402 cells by LFB;Annexin V-FITC / PI double-labeled flow cytometry was used to analyze the changes of uh-7 and Bel-7402 cells cycle ratio after being treated by different concentrations of LFB.The results showed that LFB was cytotoxic to Bel-7402 and Huh-7 in a time-dependent and concentration-dependent manner,and was not significantly toxic to QSG-7701 and L02 when treated for 72 h.The inhibition rate of LFB on the proliferation of Huh-7 and Bel-7402 cells increased with the increase of LFB concentration.It shows that LFB can selectively produce toxicity to liver cancer cells Bel-7402 and Huh-7,but has no obvious toxicity to normal liver cells in this concentration range.LFB arrested Bel-7402 and Huh-7 cell in S-phase and inhibited their proliferation activity in a concentration-dependent manner.The effect of LFB on the apoptosis rate of Huh-7 and Bel-7402 cells was analyzed by Annexin V-FITC / PI double-labeled flow cytometry;the changement of intracellular concentration of ROS when treated by LFB for 24 h at concentration on 0,0.6 mg / m L,0.8 mg / m L,and 1.0mg / of Huh-7 and Bel-7402 cells was observed by fluorescence inverted microscope,intracellular mitochondrial membrane potential and MDA and SOD levels changes were detected through the kit;5m M NAC was used to treat the cells 2h,and then the cells were treated with LFB for 24 h and was compared with the LFB-treated group to verify whether the LFB-induced apoptosis effect was caused by the increase of intracellular ROS.The results showed that with the increase of the concentration of LFB,the reactive oxygen concentration in Huh-7 and Bel-7402 cells gradually increased,and the mitochondrial membrane potential of the cells showed a downward trend.The increase of MDA content and the decrease of SOD content in the cells indicate that LFB may induce apoptosis of Huh-7 and Bel-7402 cells through the mitochondrial pathway mediated by reactive oxygen species.The results of flow cytometry showed that the apoptosis rate of Huh-7 and Bel-7402 cells in the NAC add LFB group was lower than that in the LFB alone treatment group,indicating that reducing the intracellular reactive oxygen concentration can inhibit apoptosis on Bel-7402 and Huh-7 induced by LFB.Studies have shown that LFB induces apoptosis in Bel-7402 and Huh-7 cells by activating mitochondrial apoptosis mediated by reactive oxygen species.RT-PCR and Western blot were used to verify the mechanism of the anti-tumor activity of LFB from both transcription and translation levels.The results showed that the levels of pro-apoptotic proteins Bax,Cytochrome c and Cleared-PARP in Huh-7 and Bel-7402 cells were increased,the expression of the inhibitory apoptosis protein Bcl-2 was decreased,and showed the same results at the level of m RNA and protein;Protein Cyclin E and CDK2 expression were decreased;the proliferation markers PCNA and the regulator JAK / STAT3 pathway,p-STAT3 and p-JAK2 content were decreased,indicating that LFB reduces the expression of CDK2-cyclin E complex and inhibits the activation of JAK2 / STAT3 pathway to inhibit cell proliferation.Studies have shown that LFB up-regulated the m RNA and protein content of Bax,Cytochrome c,and Cleared-PARP,reduced Bcl-2 m RNA and protein levels to activat mitochondrial apoptosis pathway mediated by reactive oxygen to induce apoptosis in Bel-7402 and Huh-7 cells.By reducing the concentration of CDK2-cyclin E complex and inhibiting JAK2 / STAT3 pathway,the proliferation activity was inhibited. |
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