title: A Hybrid Chalcone Combining the Trimethoxyphenyl and Isatinyl Groups Targets Multiple Oncogenic Proteins and Pathways in Hepatocellular Carcinoma Cells.
abstract: Small molecule inhibitors that can simultaneously inhibit multiple oncogenic proteins in essential pathways are promising therapeutic chemicals for hepatocellular carcinoma (HCC). To combine the anticancer effects of combretastatins, chalcones and isatins, we synthesized a novel hybrid molecule 3',4',5'-trimethoxy-5-chloro-isatinylchalcone (3MCIC). 3MCIC inhibited proliferation of cultured HepG2 cells, causing rounding-up of the cells and massive vacuole accumulation in the cytoplasm. Paxillin and focal adhesion plaques were downregulated by 3MCIC. Surprisingly, unlike the microtubule (MT)-targeting agent CA-4 that inhibits tubulin polymerization, 3MCIC stabilized tubulin polymers both in living cells and in cell lysates. 3MCIC treatment reduced cyclin B1, CDK1, p-CDK1/2, and Rb, but increased p53 and p21. Moreover, 3MCIC caused GSK3β degradation by promoting GSK3β-Ser9 phosphorylation. Nevertheless, 3MCIC inhibited the Wnt/β-catenin pathway by downregulating β-catenin, c-Myc, cyclin D1 and E2F1. 3MCIC treatment not only activated the caspase-3-dependent apoptotic pathway, but also caused massive autophagy evidenced by rapid and drastic changes of LC3 and p62. 3MCIC also promoted cleavage and maturation of the lysosomal protease cathepsin D. Using ligand-affinity chromatography (LAC), target proteins captured onto the Sephacryl S1000-C12-3MCIC resins were isolated and analyzed by mass spectrometry (MS). Some of the LAC-MS identified targets, i.e., septin-2, vimentin, pan-cytokeratin, nucleolin, EF1α1/2, EBP1 (PA2G4), cyclin B1 and GSK3β, were further detected by Western blotting. Moreover, both septin-2 and HIF-1α decreased drastically in 3MCIC-treated HepG2 cells. Our data suggest that 3MCIC is a promising anticancer lead compound with novel targeting mechanisms, and also demonstrate the efficiency of LAC-MS based target identification in anticancer drug development.
authors.affiliation: Department of Cell Biology, School of Basic Medical Sciences, Peking University, Beijing, China.
authors.forename: Lili
authors.initials: L
authors.lastname: Cao
authors.affiliation: School of Life Sciences, Peking University, Beijing, China.
authors.forename: Lijun
authors.initials: L
authors.lastname: Zhang
authors.affiliation: Department of Cell Biology, School of Basic Medical Sciences, Peking University, Beijing, China.
authors.forename: Xiang
authors.initials: X
authors.lastname: Zhao
authors.affiliation: Department of Cell Biology, School of Basic Medical Sciences, Peking University, Beijing, China.
authors.forename: Ye
authors.initials: Y
authors.lastname: Zhang
mesh_terms: D000970:Antineoplastic Agents / Q000737:chemistry / Q000494:pharmacology; D017209:Apoptosis / Q000187:drug effects; D001632:Bibenzyls / Q000737:chemistry; D006528:Carcinoma, Hepatocellular / Q000473:pathology*; D002453:Cell Cycle / Q000187:drug effects; D049109:Cell Proliferation / Q000187:drug effects; D002599:Chalcone / Q000737:chemistry* / Q000494:pharmacology*; D003599:Cytoskeleton / Q000187:drug effects / Q000378:metabolism; D022001:Focal Adhesions / Q000187:drug effects; D056945:Hep G2 Cells; D006801:Humans; D007510:Isatin / Q000737:chemistry*; D008113:Liver Neoplasms / Q000473:pathology*; D058990:Molecular Targeted Therapy; D015513:Oncogene Proteins / Q000378:metabolism*; D051419:Paxillin / Q000378:metabolism; D055503:Protein Multimerization / Q000187:drug effects; D020836:Protein Structure, Quaternary; D014404:Tubulin / Q000737:chemistry; D014617:Vacuoles / Q000187:drug effects / Q000378:metabolism; D060449:Wnt Signaling Pathway / Q000187:drug effects
chemical_list: D000970:Antineoplastic Agents; D001632:Bibenzyls; D015513:Oncogene Proteins; D051419:Paxillin; D014404:Tubulin; D002599:Chalcone; C040105:combretastatin; D007510:Isatin