Higenamine Enhances The Anti-proliferation Effects Of Cucurbitacin B In Breast Cancer By Inhibiting The Interaction Of AKT And CDK2

Objective:The present study aimed to reveal whether higenamine,derived from the Chinese medicine of Aconiti Lateralis Radix Praeparata can enhance the inhibitory effect of cucurbitacin B(Cu B),isolated form the Chinese medicine Trichosanthes kirilowii Maxim.on breast cancer.Furthermore,the methods of network pharmacology and western blot were used to predict and confirm the underlying mechanism.Methods:1.Anti-proliferation effect on SKBR3,T47 D of Cu B and/or higenamine at different ratio and time was assayed by CCK-8 assay kit.2.The induction of apoptosis on SKBR3,T47 D by Cu B and/or higenamine treatment in breast cancer cells was assessed by using DAPI staining and Annexin V/PI double staining.3.The effect of Cu B and higenamine on cell cycle was tested by flow cytometer.4.The mechanism of inhibitory effect of Cu B and/or higenamine on the proliferation of breast cancer was predicted by network pharmacology.5.The effect of Cu B and/or higenamine on the expression of AKT,p-AKT,CDK2,p-CDK2,cyclin A2,Bax,Bcl-2 and p-Histone H3 was checked by western blot.6.SKBR3 nude-mouse transplanted tumor model was established to observe the inhibitory effects of Cu B and/or higenamine on breast cancer in vivo.Results:1.Higenamine enhanced the inhibitory effects of Cu B on breast cancer cells.The growth of SKBR3 and T47 D cells was significantly inhibited by Cu B in a concentration-and time-dependent manner.Cu B: higenamine at the ratio of the10:1 resulted in the strongest inhibitory effect on cell growth among the ratios designed in this experiment.2.Cu B can induce apoptosis,and higenamine can enhance the percentage of apoptotic cells.DAPI staining showed that the combination of Cu B and higenamine clearly increased nuclear chromatin condensation and granular apoptotic bodies.As shown in the result of flow cytometric,the percentage of apoptotic cells in the group of combined Cu B and higenamine treatment was higher than that of single Cu B or higenamnie treatment in both SKBR3 and T47 D cell lines.3.Cu B induced G2/M phase arrest and the combined treatment of Cu B and higenamine resulted in a higher accumulation of cells in the G2/M phase compared with the treatment of Cu B or higenamine alone in both the SKBR3 and T47 D cell lines.4.SKBR3 xenografts demonstrated that the combination treatment drastically and effectively suppressed tumor growth compared with Cu B or higenamine alone.5.Network pharmacology revealed that AKT1 was thought to be the major target of Cu B in breast cancer,and the targets of higenamine were cyclin A2 and CDK2.Downstream effector protein of AKT1 is CDK2 and cyclin A2.Cu B inhibits phosphorylation of AKT1 and also inhibits phosphorylation of CDK2,while higenamine acts on CDK2 and cyclin A2 to enhance this action of Cu B.6.The combination of Cu B and higenamine resulted in the down-regulation of p-AKT,p-CDK2,cyclin A2 and Bcl-2,up-regulation of Bax and p-Histone H3 compared with administration of agent alone.And levels of AKT and CDK2 showed no changes among groups.Conclusions:1.Cu B can inhibit growth of breast cancer cell lines,and higenamine can enhance this effect by anti-proliferation,cell cycle arrest and induction of apoptosis.2.Cu B combined with higenamine has synergistic anti-breast cancer effect in SKBR3 xenograft.3.Network pharmacology revealed that Cu B and higenamine may synergistically inhibit breast cancer cell proliferation by inhibiting the interaction between AKT and CDK2.