Norcantharidin inhibits DNA replication and induces apoptosis with the cleavage of initiation protein Cdc6 in HL-60 cells.
Norcantharidin (NCTD), a demethylated form of cantharidin, is currently used as an anti-cancer drug in China. However, the exact anti-cancer mechanism of NCTD on human cancer cells remains poorly understood. In the present study, NCTD inhibited proliferation and DNA replication effectively in HL-60 cells. DNA replication-initiation protein Cdc6 was cleaved after 12 h treatment with NCTD. This cleavage generated a truncated Cdc6 fragment with a relative molecular weight of 49 kDa and elongated treatment with NCTD resulted in a complete loss of Cdc6. In addition, we found that Cdc6 was present in both non-chromatin- and chromatin-bound fractions in the untreated HL-60 cells, and NCTD treatment led to the cleavage of Cdc6 in both fractions. NCTD-induced cleavage of Cdc6 was prevented by pre-treatment with caspase-3 inhibitor, suggesting the involvement of caspase-3 activity in the process. Furthermore, NCTD treatment resulted in apoptotic changes including granular nuclear morphology, DNA laddering and sub-G1 arrest in HL-60 cells. In conclusion, our study reveals that NCTD can inhibit DNA replication, and induce apoptosis and caspase-3-dependent cleavage of Cdc6. The anti-cancer effect of NCTD may be closely associated with the dysfunction of Cdc6 and our report is the first to put forward this point of view.
Li
Jin-Long
JL
State Key Laboratory of Oncology in Southern China.
Cai
Yu-Chen
YC
Liu
Xu-Hui
XH
Xian
Li-Jian
LJ
D017209:Apoptosis / Q000187:drug effects* / Q000502:physiology; D019086:Bridged Bicyclo Compounds, Heterocyclic / Q000494:pharmacology*; D053148:Caspase 3 / Q000378:metabolism; D018797:Cell Cycle Proteins / Q000378:metabolism*; D004261:DNA Replication / Q000187:drug effects*; D004273:DNA, Neoplasm / Q000096:biosynthesis; D018922:HL-60 Cells; D006801:Humans; D009687:Nuclear Proteins / Q000378:metabolism*; D013936:Thymidine / Q000378:metabolism; D014316:Tritium
D019086:Bridged Bicyclo Compounds, Heterocyclic; C103706:CDC6 protein, human; D018797:Cell Cycle Proteins; D004273:DNA, Neoplasm; D009687:Nuclear Proteins; D014316:Tritium; C069741:norcantharidin; D053148:Caspase 3; D013936:Thymidine