Studies On The Anti-proliferative Effects And Molecular Mechanism Of Ailanthone In Hepatocellular Carcinoma

Aim: To investigate anti-hepatocellular carcinoma activity of ailanthone and elucidate its antitumor molecular mechanisms.Methods:(1) The growth inhibitory activity of ailanthone on Hep G2, Hep3 B and Huh7 cell lines was detected by MTT assay;(2) The influence of ailanthone on long-term cell proliferation was observed by cell cloning experiment method;(3) The influence of ailanthone on cell cycle was detected by PI;(4) The effect of ailanthone on DNA damage was performed by single cell gel eletrophoresis;(5) Fluorescence intensity of p-H2 AX was evaluated by immunofluorescence assay;(6) The apoptosis rate was detected using Annexin V-FITC/PI;(7) The mitochondrial membrane potential was detected using JC-1 flow cytometry assay;(8) The expression levels of the main proteins that regulate the cell cycle, apoptosis, mitochondria apoptosis pathway and Akt were detected by Western blot;(9) Anti-hepatocellular carcinoma activity of ailanthone in vivo was evaluated in Huh7 tumor xenografts;(10) Pathological changes of organs and relative proteins changes in vivo were observed on HE staining and immunohistochemical staining.Results: Ailanthone showed obvious cytotoxicity to Huh7 in a time- and dose-dependent manner in vitro, and it could inhibit the long-term proliferation of Huh7. Flow cytometric analysis demonstrated that ailanthone could induce cells arrest in G0/G1 phase. Western blot results showed that expression level of the key proteins that regulating cell cycle phase, Cyclin D, Cyclin E, CDK2, CDK4, CDK6, CDC25 A, and Rb was downregulated; while p21 and p27 was upregulated. In addition, ailanthone triggered DNA damage through DNA double stranded breaks, and subsequently leading to the phosphorylation and activation of ATR/ATM. Annexin V-FITC/PI and JC-1 staining method shown that, ailanthone could induce cells apoptosis, along with decrease in mitochondria membrane potential. Apotosis-inhibitory agent Z-VAD-FMK, could partially reduce the toxic effects of ailanthone on Huh7 cells. Cyto c was released from the mitochondria into cytoplasm, Bcl-2 was downregulated, Bax was upregulated, and Bax was turned into mitochondria from cytoplasm after ailanthone treatment. The levels of phosphorylation of Akt protein(Ser473 and Thr308) were decreased after ailanthone treatment. In vivo studies demonstrated that ailanthone potently inhibited the growth of nude mice bearing Huh7 xenografts. No obvious toxicity towards heart, liver, spleen, lung, and kidney has been observed at the level of the effective does in ailanthone treatment groups. Immunohistochemical study showed that ailanthone can induce cells apoptosis, downregulate CDK4, and destroy the tumor blood vessels in vivo.Conclusion: Ailanthone has potent inhibition effect on liver cancer Huh7 cell growth in vitro. Ailanthone induces G1/S cell cycle arrest through DNA double-stranded damage and the regulation of cell cycle key proteins. Ailanthone can downregulate the expression of Bcl-2 protein family, and induce cell apoptosis by the mitochondrial apoptosis pathway. Ailanthone can inhibit the PI3K/AKT signal pathway of abnormal activation. Ailanthone also has the anti-hepatocellular carcinoma activity in vivo.