Purification Of An Anticancer Protein From Hemerocallis Citrine Baroni And Its Mechanism Of Inhibiting The Proliferation In Human Hepatocarcinoma Cells

Hepatocellular carcinoma(HCC)is a common malignant epithelial cell tumor.Its incidence is related to cirrhosis of the liver,alcoholism,genetic mutation,virus infection and chemical carcinogens.At present,the main treatment methods for liver cancer include surgical treatment and chemotherapy,but these methods have great toxic and side effects on human body,so their clinical application is largely limited.Recent research reports have shown that proteins extracted from some plants have not only anti-tumor activity,but also low toxicity and side effects,which will provide new ideas and insights for the extraction of chemical substances from natural products in the field of biology and medicine.Hemerocallis citrina Baroni is a perennial perennial root herb of the Liliaceae Hemerocallis genus.Hemerocallis citrina Baroni is rich in nutrition,containing protein,sugar,vitamins,polyphenols,terpenes,anthraquinones,steroids,saponins,alkaloids and other biological active substances,with tranquilizing,hemostasis,anti-inflammatory and anti-oxidation,digestion,heat,anti-depression effect,so Hemerocallis citrina Baroni is a kind of medicine and food homology material.It has been reported that the extract of Hemerocallis citrina Baroni has anticancer activity,but the anti-tumor activity of the protein extracted from Hemerocallis citrina Baroni is little studied.Therefore,in this study,an anti-tumor active protein was isolated and purified from Hemerocallis citrina Baroni,and the mechanism of its inhibition of HCC cell proliferation was studied.The main research contents include the following three parts:The first part: Isolation and purification of antitumor protein from Hemerocallis citrina Baroni.Combined with cell survival assay(MTT assay),the crude protein are extracted by using phosphate buffer extraction and acetone precipitation.An anti-tumor active protein named HcBP was obtained by further saturated ammonium sulfate precipitation,and twice cation exchange chromatography.Normal liver cells(HL7702)and liver cancer cells(HepG2 and Bel-7402)were treated with different concentrations of HCBP,so as to detect the effect of HCBP on the proliferation of liver hepatocellular carcinoma.The results of cell survival assay(MTT)showed that HCBP could significantly inhibit the survival activity of liver cancer cells,but had no significant effect on the survival activity of normal liver cells.The effect of HCBP on HepG2 and Bel-7402 cells was further detected by cell clone formation assay.The results showed that HCBP could significantly inhibit the clone formation ability of HepG2 and Bel-7402 cells.These results indicate that HcBP is an anti-tumor active protein with low toxicity effects.The results of mass spectrometry and UV scanning showed that the protein was similar to the peroxidase in Arabidopsis thaliana,and contained heme progroup.The peroxidase activity of HcBP was detected by the peroxidase activity.The second part: HCBP induces the apoptosis of hepatocellular carcinoma cells through mitochondria-mediated pathway.DAPI staining and flow cytometry were used to detect the effect of HCBP on the apoptosis of HepG2 and Bel-7402 cells,and the results showed that HCBP could significantly inhibit the apoptosis of HepG2 and Bel-7402 cells.Finally,the effects of HCBP on ROS levels and key proteins in apoptosis were detected.The results showed that ROS levels in HepG2 and Bel-7402 cells were significantly increased after HCBP treatment.In addition,Western blotting results showed that the protein levels of Activate-Caspase-9,Activate-Caspase-3,Cytochrome C and Bax were significantly up-regulated after HCBP treatment.The protein level of Bcl-2 was significantly down-regulated.These results indicated that HCBP inhibited the apoptosis of HepG2 and Bel-7402 cells through the mitochondrial apoptosis pathwayThe third part: HCBP inhibits the aerobic glycolysis of HCC cells by regulating PKM2.HepG2 and Bel-7402 cells were treated with different concentrations of HcBP,and the cell culture medium was collected to detect grape culture residual amount and lactic acid production in the culture medium.The results showed that after HcBP treatment of the two hepatoma cells,the glucose residual amount in the culture medium increased significantly,while the lactic acid production decreased significantly.And then real-time fluorescent quantitative PCR method was used to detect HcBP ACTS on the key proteins related to the aerobic glycolysis in the liver cancer cells.The results show that the HcBP significantly reduced the GLUT1,LDHA and PDK m RNA level.Next,Western blotting was used to detect the changes in the protein level of pyruvate kinase(PKM2),and the enzyme activity of pyruvate kinase(PKM2)was detected.The results showed that HcBP treatment of two hepatocellular carcinoma cells not only significantly reduced the total cell PKM2 protein level,but also significantly reduced the PKM2 protein level in the nucleus and cytoplasm.After over expressing PKM2,the effects on glucose consumption,lactic acid,PKM2 and the aerobic glycolysis key protein were detect.The results show that a marked increase in the residual amount of glucose in the culture medium,and the decrease of lactic acid production obviously got remarkable reversal,after PKM2 expression.In addition,the expressions of the key proteins related to the aerobic glycolysis GLUT1,LDHA and PDK also be increased.Therefore,these results suggest that HCBP inhibit aerobic glycolysis of HepG2 and Bel-7402 cells by regulating PKM2.In summary,in this study,an anti-tumor active protein named HcBP with low toxicity was isolated and purified from Hemerocallis citrina Baroni.Studies have shown that HCBP can significantly inhibit the proliferation of HCC cells and induce the apoptosis of HCC cells.In addition,HCBP can inhibit the aerobic glycolysis of HCC cells by regulating PKM2.