Inhibitory Effect On Proliferation Of Cancer Cells And The Mitochondrial Mechanism Of Both Asiatic Acid And Its Complexes With Rare Earth

The purpose of this study is to investigate the antitumor activity and mitochondrial mechanism of asiatic acid(AA) and AA-Rare earth complexes, simultaneously establish a transgenic yeast model to screen antitumor drugs which can induce cell death mainly via mitochondria mediated apoptosis pathway.The anti-oxidantive activities of AA and its rare earth complexes in vitro were measured by hydroxy radical-scaveng test,the effect of AA and AA-rare earth complexes(AA-Ce,AA-Tb and AA-Eu) on proliferation of human hepatoma carcinoma cell line(HepG2),human melanoma cell line(B16),human cervical carcinoma cell line(Hela),human esophageal carcinoma cell line(Eca109),human lung adenocarcinoma cell line(A549) and human breast carcinoma cell line (MCF-7) were tested by MTT,meanwhile morphology of tumor cells was observed under inverted microscope;Intracellular reactive oxygen species(ROS) content was detected with DCFH-DA,intracellular ATP level was measured by ATP Assay Kit,mitochondrial membrane potential was detected with fluorescent probe JC-1,and the expression of voltage-dependent anion channel(VDAC) on mitochondrial out membrane was determined by RT-PCR and Western Blot;DNA fragmentation was determined by agarose gel electrophoresis;the oxygen consumption rate of mitochondrial was determined by Clark oxygen electrode;and Ca²⁺-induced mitochondrial swelling model was used to observe the direct effect of AA,AA-Tb and AA-Eu on mitochondria.In order to approach the interaction between AA and VDAC,we compare the effects of AA on proliferation of transgene yeasts(the wild type Saccharomyces cerevisiae M-3 and the VDAC1 deficient type M22-2) by monitoring OD₆₀₀ and counting cell number, The results suggested that AA inhibited the proliferation of tumor cell lines dose-dependently in which HepG2 was more sensitive to AA,and cells rounded and shrank after treated with 100μmol/L AA for 24h.Mitochondrial membrane potential depolarized,intracellular ATP level dose-dependent decreased and ROS content dose-dependently increased afer treated with AA,and VDAC expression was up-regulated by 50μmol/L AA;Furthermore,isolated mitochondria were dose-dependently protected by AA against swelling induced by Ca²⁺,which suggested AA can react on mitochondria directly.The main active groups of AA-rare earth complexes are the same,but presented higher inhibitory effects on Eca109,Hela,A549 and MCF-7;Oxygen consumption rate decreased,and mitochondial swelling induced by Ca²⁺ was inhibited by treatment of AA-Tb or AA-Eu on Hela cells.The proliferation of M-3 and M22-2 yeast were inhibited by 5-Fu with no selectivity,suggesting VDAC was not invovled in cytotoxicity of 5-Fu.As₂O₃ and AA had conspicuous inhibitory effects on proliferation of M-3 but not of M22-2, illustrating that As₂O₃ and AA induced yeast apoptosis by interaction with VDAC.In conclusion,both AA and AA-rare earth complexes have good antitumor effects,which was related with mitochondria mediated apoptosis,and VDAC was involved in the pathway;Meanwhile a new platform to study the mechanism of antitumor drugs via mitochondrial-mediated apoptosis was established.