Inhibition Effects And Mechanisms Of Berberine On HepG2Hepatoma Cell Proliferation And Repopulation

Cancer epidemiology research shows that Hepatocellular Carcinoma is a threatdisease to human being with high mortality. At present, radiotherapy, chemotherapyand surgery is the main method of treatmen, but the recurrence rate is vitalshortcoming. Therefore, finding effective treatments and preventing recurrence areconsiderable problems to be solved.Reports show that caspase-3can induce cell apoptosis and promote cellproliferation at the same time. Activated caspase-3is the executor of the apoptosis,but also can stimulate the growth of cell proliferation. Caspase-3is the key factor ofrecurrence which is induced by dead tumor cells. The abnormality of arachidonicacid metabolism is one of the important factors of tumor, cPLA₂and COX-2are twokey enzymes in arachidonic acid metabolic pathways. The membrane phospholipidsare converted to AA by cPLA₂, and then AA is catalyzed into PGE₂by COX-2.Activated caspase-3can activate the PLA₂to start the arachidonic acid metabolicpathway, eventually leading to the release of PGE₂. PGE₂can inhibit tumor cellapoptosis and promote tumorigenesis and metastasis, the antiapoptotic effect ofPGE₂is the direct factor of tumor recurrence. Above all, caspase-3-PLA-PGE₂signaling pathways link tumor cell apoptosis to proliferation. The release of PGE₂induced by activated caspase stimulates proliferation of few survival tumor cells andleads to tumor recurrence. The dead cells stimulating proliferation of survival cellsmeans that the treatment strategy of inducing tumor cell apoptosis is faced withenormous challenges. Berberine, a kind of Chinese medicinal herb, has many advantages,such as smallside effects and wide pharmacological effects. A great many medical researchers payattention on the anticancer activity of berberine in recent years, but its inhibition oftumor recurrence has not been reported. Berberine can induce tumor cells apoptosis incaspase independent way, suggesting that berberine can inhibit tumor growth andavoid tumor repopulation at the same time.Objective:Berberine on HepG2cell proliferation and recurrence are studied in this paper.Westudied the influence of berberine on the regulation of caspase and arachidonic acidmetabolism in correlation with tumor apoptosis and recurrence, and the inhibitoryeffect and mechanism of berberine on liver cancer and recurrence.It will providetheoretical basis for the choice of clinical treatment of liver cancer drugs.Method and Result:1. Inhibition Effects and Mechanisms of Berberine on HepG2Hepatoma CellProliferationHepG2cell are treated with berberine of different concentrations （0,12.5,25,50,100microns） for24,48and72h, determined by MTT method to detect cellproliferation inhibition of berberine; Flow cytometry instrument to detect apoptosisinduced by berberine; Western blot to detect the expression of caspase-3, caspase-9,cPLA₂, COX-2and AIF; PGE₂and AA concentrations in the supernatant weremeasured by ELISA.Results showed that berberine can inhibit proliferation of HepG2cells, dosing cellsurvival rate is lower than the control group （P <0.05）, and in time and dosedependent manner, the IC50value at72h was82.8μM; flow cytometry analysisshowed that BBR increased the number of late apoptotic cells （P <0.05）; comparedwith control group, each dose berberine had no effect on the expression and activityof caspase-9and caspase-3; a wide range of BBR from12.5μM to100μM inducedsignificant increase in the expression of nucleic AIF protein （P <0.05）,and significantdecrease in the expression of cPLA₂and COX-2（P <0.05）; the content of AA waselevated with the increase of BBR dosage（P <0.05）, the level of PGE₂was significantly reduced even when in a low dose of BBR （12.5μM）（P <0.05）,the ratioof AA to PGE₂concentration was increased after BBR treatment（P <0.05）.2. Inhibition Effects and Mechanisms of Berberine on HepG2Hepatoma CellRepopulationBased on the tumor recurrence model induced by VP-16, determined by MTTmethod to detect inhibitory effect of berberine on recurrence; Western blot to detectthe expression of caspase-3, cPLA₂, and cox-2; Enzyme catalyzing the substrate thekits for caspase3activity; ELISA to detect the content of AA and PGE₂.Results showed that berberine can inhibit the recurrence of HepG2cells inducedby VP-16, for cell survival rate of dosage group is lower than the model group （P <0.05）; compared with model group, berberine inhibits protein expression and activityof caspase-3in the feeder cells （P <0.05）; compared with model group, berberineinhibits protein expression of cPLA₂and COX-2in the feeder cells （P <0.05）; thecontent of AA was elevated with3.125uM BBR （P <0.05）, the level of PGE₂wassignificantly reduced with3.125uM BBR （P <0.05）,the ratio of AA to PGE₂concentration was increased after BBR treatment（P <0.05）.Conclusion:The apoptosis induced by berberine in HCC is AIF-mediated and caspase-independent,and the elevated ratio of AA to PGE₂by suppressing cPLA₂and COX-2pathways isinvolved in the inhibitory effect of berberine on HCC; berberine shows the inhibitoryeffect on HCC repopulation, through suppressing arachidonic acid metabolic pathwayand the release of PGE₂.