In Vitro Evaluation Of The Anticancer Effects Of Berberine Hydrochloride And D-limonene Combination On Human Gastric Carcinoma Cell Line MGC-803

Purpose:In order to evaluate the anticancer effects of berberine hydrochloride (berberine for short) and d-limonene(limonene for short) alone and in combination on human gastric carcinoma cell line MGC-803,and define their mechanisms of action.Methods:MGC-803 cells were treated with berberine and limonene alone and in combination for 24 to 48 hours.Inhibitory effects on growth were determined by the MTT assay.The combination index(CI) and drug reduction index(DRI) were calculated with the computerized Chou-Talalay method which is based on the median-effect principle. Flow cytometry and laser scanning confocal microscope were employed to evaluate the effects on cell cycle perturbation and apoptosis,generation of reactive oxygen species, mitochondrial membrane potential,and the expression of Bcl-2,Caspase-3 proteins in MGC-803 cells.Results:1.Both berberine and limonene preferentially inhibit the growth of MGC-803 cells in a dose-effect relationship.Based on the antiproliferative D_m values after incubation with drug for 48 h,the combination of berberine hydrochloride and d-limonene(1:4) ratio exhibited synergistic inhibition of MGC-803 cells when treated with drug combination 0.1～1 mM for 24 h,and 0.04～0.1 mM for 48 h,combinations allowed for a dose reduction of 1.8～3.8 fold for berberine and 3.6～5.4 fold for limonene as indicated by the dose reduction index(DRI),and combination index(CI)＜1.2.Cell cycle analysis indicated that berberine blocked cells in G₀/G₁ phase,whereas limonene blocked cells in G₀/G₁ and G₂/M phase.Interestingly,drug combination exposure led to accumulation of cells in the G₀/G₁ and G₂/M phase,a pattern similar to that caused by limonene alone.The induction rate of apoptosis for 36 and 48 h by drug combination treatment(26%and 29%respectively) was significantly greater than that of drug used alone(p＜0.05),whereas the rate being not different from those induced by limonene singly when treated for 24 h.3.Berberine and limonene alone and in combination could significantly induce intracellular ROS generation,reduce mitochondrial transmembrane potential△ψm, enhance expression of Caspase-3 and decrease expression of Bcl-2,combination therapy of those two drugs showed more remarkable effects when compared with drug used singly in MGC-803 cells. Conclusion:1.Berberine and limonene used alone and in combination could inhibit the growth of MGC-803 cells effectively.The combination of them showed synergistic effect at a certain range of dose.2.Limonene might have a dominant effect in cell cycle progression as compared to berberine when combined exposure.Combining those two drugs induced apoptosis of MGC-803 cells,and more time was needed to increase the rate of apoptosis for combination therapy showed more remarkable effects when compared with limonene used singly.3.Induction of ROS generation,mitochondrial dysfunction and regulation of apoptosis related proteins demonstrate that combination agents exert antiproliferative effects against MGC-803 cells that is mediated through apoptosis.