ABSTRACT
Aim To explore the relationship between Mre11/Rad50/Nbs1 ( MRN ) complex focus formation and DNA double-strand breaks( DSBs) caused by cinob-ufagin in human hepatocellular carcinoma HepG2 cells. Methods The Na+,K+-ATPaseα1 subunit expression level in liver cancer tissues was detected by immunohis-tochemistry. After HepG2 cells were treated with 5μmol·L-1 cinobufagin for 6, 12 and 24 h, the drug-in-duced DSBs were assessed by single cell gel electro-phroesis ( SCGE ) , the gene transcription and protein levels of Mrel1, Nbs1, Rad50 and p53 were evaluated by Real time-PCR and Western blot. The cell cycle in parallel was analyzed by flow cytometry. Results The Na+, K+-ATPase α1 subunit expression level in liver cancer tissues was significantly increased compared with the tissue adjacent to carcinoma ( P <0. 05 ) . The 5μmol · L-1 cinobufagin could induce the DSBs in a time-dependent manner (P <0. 05), and it could up-regulate the gene expression levels of Mre11, Nbs1, Rad50 and p53 in HepG2 cells ( P<0. 05 ) . The pro-portions of HepG2 cells in S phase were ( 21. 32 ± 4. 21) % in the control group, and (33. 25 ± 5. 72) %, (56. 72 ± 6. 29) % and (67. 32 ± 9. 42) % in HepG2 cells treated with 5 μmol · L-1 cinobufagin for 6, 12 and 24 h, respectively. The proportions of cells in S phase in cinobufagin groups were significantly increased compared with the control group ( P<0. 05 ) . Conclu-sion Cinobufagin could induce the cell cycle arrest in liver cancer HepG2 cells by activation of Mre11/Rad50/Nbs1 Complex.
ABSTRACT
Aim To investigate the effect of ouabain and cinobufogenin on cell proliferation,apoptosis and cell cycle on HepG2,and explore their molecular mechanism.Methods The anti-proliferative effect on HepG2 cells was determined by MTT assay.The HepG2 cells were stained with Hoechst 33342,and its morphological changes were observed under fluorescence microscope;The cell cycle was measured by flow cytometry.The Cyclin A1,CDK 2,PCNA and p21~(CIP1) expression levels of HepG2 cells treated with ouabain and cinobufogenin were dectected in mRNA and protein by Real time PCR and Western blot.Results Ouabain and cinobufogenin could inhibit cell proliferation on HepG2 cells,and the inhibitory effects were in time and dose dependent manners.The HepG2 cells treated with ouabain and cinobufogenin showed the typical morphological features of apoptosis.Cell cycle analysis showed that the S phase of HepG2 cells treated with ouabain and cinobufogenin increased significantly compared with the control group.Real-time quantitative PCR and Western blot results showed that ouabain and cinobufogenin could down-regulate Cyclin A1,CDK 2,and PCNA expressions(P<0.05)and up-regulate p21~(CIP1) expression(P<0.05).Conclusion Nα~+,K~+-ATPase inhibitor has the anti-proliferative effect on HepG2 cells and induce apoptosis and S phase arrest.These effects might be related with proteins associated with cell cycle closely.