title: Norcantharidin inhibits pre-replicative complexes assembly of HepG2 cells.
abstract: Norcantharidin (NCTD) is currently used for anticancer therapy but the exact mechanism of action remains unknown. Pre-replicative complexes (pre-RCs) are essential for cell DNA replication and highly related to malignant proliferation. Here, we examined the inhibitory effect of NCTD on pre-RC components in HepG2 cells. We showed that NCTD induced degradation of Cdc6 and Mcm2 in a dose-dependent manner. Under 100 μM NCTD concentration, about 70% of Cdc6 and 50% of Mcm2 were degraded. In addition, the nuclear translocation of Mcm6 was inhibited by NCTD. Further studies aiming at G1 synchronous cells showed that, NCTD reduced the chromatin-bound Cdc6, Mcm2 and Mcm6. Moreover, the cells were blocked from entering the S phase and accumulated at the G1 phase when released synchronously into the cell cycle. Consistently, the DNA replication was inhibited by NCTD. Finally, the combination NCTD with Cdc6 depletion lead to more severe cytotoxicity (88%) than NCTD (52%) and Cdc6 depletion (39%) alone. A synergic cytotoxicity was observed between Cdc6 depletion and NCTD. In conclusion, our results demonstrate that NCTD inhibits pre-RC assembly; subsequently blocks the G1 to S transition; and inhibits DNA replication in HepG2 cells. Pre-RCs are an intriguing target for cancer therapy, which merits further investigations for anticancer development.
authors.affiliation: Institute of Biotherapy, School of Biotechnology, Southern Medical University, Guangzhou, China.
authors.forename: Sansan
authors.initials: S
authors.lastname: Chen
authors.forename: Xinming
authors.initials: X
authors.lastname: Qu
authors.forename: Pei
authors.initials: P
authors.lastname: Wan
authors.forename: Qing Wen
authors.initials: QW
authors.lastname: Li
authors.forename: Ziyi
authors.initials: Z
authors.lastname: Wang
authors.forename: Fangfang
authors.initials: F
authors.lastname: Guo
authors.forename: Li
authors.initials: L
authors.lastname: Bai
authors.forename: Zhiming
authors.initials: Z
authors.lastname: Hu
authors.forename: Wanlong
authors.initials: W
authors.lastname: Tan
authors.forename: Jinlong
authors.initials: J
authors.lastname: Li
mesh_terms: D000970:Antineoplastic Agents / Q000494:pharmacology / Q000627:therapeutic use*; D001692:Biological Transport; D019086:Bridged Bicyclo Compounds, Heterocyclic / Q000494:pharmacology / Q000627:therapeutic use*; D002193:Cantharidin / Q000031:analogs & derivatives / Q000494:pharmacology / Q000627:therapeutic use; D002453:Cell Cycle / Q000187:drug effects*; D018797:Cell Cycle Proteins / Q000378:metabolism*; D002467:Cell Nucleus / Q000187:drug effects; D002843:Chromatin / Q000378:metabolism; D004261:DNA Replication / Q000187:drug effects*; D056945:Hep G2 Cells; D006801:Humans; D008113:Liver Neoplasms / Q000188:drug therapy* / Q000235:genetics / Q000378:metabolism; D064111:Minichromosome Maintenance Complex Component 2; D064151:Minichromosome Maintenance Complex Component 6; D009687:Nuclear Proteins / Q000378:metabolism*
chemical_list: D000970:Antineoplastic Agents; D019086:Bridged Bicyclo Compounds, Heterocyclic; C103706:CDC6 protein, human; D018797:Cell Cycle Proteins; D002843:Chromatin; D009687:Nuclear Proteins; C069741:norcantharidin; C084859:MCM2 protein, human; C103258:MCM6 protein, human; D064111:Minichromosome Maintenance Complex Component 2; D064151:Minichromosome Maintenance Complex Component 6; D002193:Cantharidin