Synergistic Anti-glioma Effect Of Hydroxygenkwanin And Apigenin In Vitro

Objective: To investigate synergistic anti-glioma effects of Hydroxygenkwanin(HGK) and Apigenin (AP) in vitro, meanwhile study the possible mechanisms.Methods: The time-and dose-dependent anti-proliferation manner of HGK, AP,AP+HGK on C6glioma cell line was firstly evaluated by MTT assay. After treatment,acridine orange/ethidium bromide (AO/EB) staining and4’,6-diamidino-2-phenylindole (DAPI) staining were applied to assess the apoptotic morphology changein each group. The structural and functional change of mitochondria was separatelyassessed by transmission electron microscopy (TEM) and JC-1fluorescence probes(JC-1), a commonly used method for detecting mitochondrial membrane potential (MPP)loss. Single cell gel eletrophoresis (SCGE) and flow cytometric analysis wereconducted to investigate the DNA damage and cell cycle arrest effect in each treatedgroup. Additionally, western blot was also applied to investigate the expressions ofsome typical anti-, pro-apoptotic bcl-2family proteins including Bcl-2, Bcl-xl, Bax, Bak,Bid, Bad; TNF-a level and caspase-3,-8activations were also measured byradioimmunoassay kit (RIA kit) and correlated caspase activity assay kits.Results: The experimental results showed that both HGK and AP inhibits C6glioma proliferation in an dose and time dependent manner, HGK exhibits strongeranti-glioma effects compare to AP at the same concentration and during the sametreating time. HGK and AP present dramatic synergistic anti-glioma effects when theywere administrated simultaneously. AO/EB staining and DAPI staing indicated that HGK and AP have the function of apoptosis inducing effect on C6glioma cells. MMPloss and mitochondrial damage were oberved by TEM and JC-1in each treated groupand AP+HGK show the most severe mitochondria damage, so as the results of DNAdamage obseved in SCGE. The results of flow cytometric analysis detected the cellcycle S phase arrest effect of HGK, while no such effect of AP was found in tested drugconcentration. TNF-α activation, casepase-3,8activation were observed in furthermolecular mechanisms investigation. No synergistic effects on bcl-2family proteinsexpression of HGK and AP were found in western blot. Additionally, HGK showed lowtoxicity on PC12cells (IC50>120μM)Conclusion: It is the first time that the anti-glioma effect of HGK investgated, aswell as it synergistic anti-proliferation effects when combined used with AP. All thedata suggest that HGK may be another promising anti-glioma agent, the combinedadministration of HGK and AP may be an effective method for glioma chemotherapy.The results provide useful information for further investigation of the anti-gliomaeffects of HGK and AP in vivo.