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The Antitumor Activity and the Underlying Mechanism of the Novel Hypoxia-activated Prodrug Q6

Author: LiuXiaoWen
Tutor: YangBo; HeQiaoJun
School: Zhejiang University
Course: Pharmacology
Keywords: hypoxia-activated prodrug Q6 Topo Ⅱ HIF-1α Autophagy-lysosomepathway hepatocellular carcinoma anti-cancer activity
CLC: R965
Type: PhD thesis
Year: 2013
Downloads: 43
Quote: 0
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Abstract


Objective:The hypoxia-activated prodrugs are the first class of drugs specifically targeting tumor hypoxia and Tirapazamine (TPZ) is one of the leading compounds. TPZ is converted by intracellular reductase(s) to a cytotoxic radical that under hypoxic conditions generates base damage to the hypoxic cells. However, when oxygen is present, the TPZ radical is back-oxidized to the nontoxic parent compound, thereby decreasing the cytotoxicity. Up to now, though many in vitro and in vivo studies have demonstrated the anti-cancer activity of TPZ, its poor lipid solubility, limited efficacy and extensive toxicity have restricted its further application. Therefore, we have screened the anti-cancer activity of many series of TPZ derivatives, in order to find novel hyxpoxia-activated prodrug which has better cancer-killing effects and less drug toxicity and also to evaluate the underlying mechanisms involved in.Methods:(1) MTT method was used to detect anti-proliferation activity of chemotherapeutic agents on different sources of human cancer cells and normal liver cells.(2) Molecular docking assay was employed to determine the Topo Ⅱ kinase activity.(3) TARDIS assay was used to detect the formation of Topo Ⅱ-DNA complex.(4) Western blotting assay were used to detect the protein expression.(5) Analysis of apoptosis and cell cycle was determined by propidium iodide staining or Annexin V/PI staining.(6) Mitochondrial membrane depolarization was determined by JC-1staining.(7) Xenografted athymic mice model and H22murine hepatoma model were introduced to investigate the in vivo anti-cancer activity of chemotherapeutic agents.(8) Real-Time reverse transcription was used to detect the mRNA level.(9) Luciferase reporter gene assay was used to detect the transcription activity of transcription factor.(10) Immunoprecipitation was used to detect the interaction between protein and protein.(11) Small interfering RNA was introduced to silence related gene.(12) Immunofluorescence analysis was used to detect the distribution of protein in cells.(13) Electron microscopy assay was employed to detect the subcellular structures.(14) immuno-transmission electron microscopy was introduced to detect the distribution of protein in subcellular structures.(15) ELISA and fluorescence polarization assay were used to determine the AKT, EGFR and PI3K kinases activity.(16)Immunohistochemical analysis were used to detect the protein distribution in tumor issues.Results:(1) Anti-tumor activity and therapeutic target of the novel hypoxia-activated prodrug Q6:The novel hypoxia-activated prodrug Q6showed broad anti-proliferation activity and hypoxia selectivity in ten different sources of human solid cancer cell lines and leukemia cell lines; moreover, Q6expressed better anti-cancer activity and hypoxia selectivity than TPZ in three hepatocellular carcinoma cell lines; two normal liver cell lines were resistant to Q6. Meanwhile, compared to TPZ, Q6has been evaluated as a more potent Topo Ⅱ inhibitor which could dramatically inhibit Topo Ⅱ-mediated kDNA decatenation, trap and stabilize Topo Ⅱ-DNA complex. Moreover, as a Topo Ⅱ inhibitor, Q6could induce DNA DSBs-mediated G2/M phase arrest and apoptosis in two hepatocellular carcinoma cell lines. Furthermore, Q6administration significantly inhibited growth of Bel-7402xenograft tumors and H22murine hepatoma.(2) The regulatory mechanism of the novel hypoxia-activated prodrug Q6on HIF-1α:Treatment with Q6markedly downregulated HIF-1α expression and transcription of the downstream target gene in a dose-dependent manner. Furthermore, Q6downregulated HIF-1α protein expression level through accelerating its degradation, rather than its transcription level and protein synthesis. Intriguingly, it is the autophagy-lysosome degradation pathway that plays a crucial role in Q6-induced attenuation of HIF-1α expression, rather than the proteasome-dependent pathway, and P62was involved in the process.Conclusion:(1) The novel hypoxia-activated prodrug Q6exhibited a more potent anticancer activity than TPZ both in vitro and in vivo; Q6was evaluated as a Topo Ⅱ inhibitor and could induce DNA DSBs-mediated cell cycle arrest and apoptosis.(2) Q6could inhibit HIF-1α protein level through accelerating autophagy-lysosome degradation pathway, and P62is involved in the process.

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