These results indicate that treatment with 300 nM UCN 01 induced arrest in S phase and G2M phases in all three cell lines. UCN 01 induces cell cycle arrest in hepatoma cells via the p53pP21waf1 and CHK2CDC25C pathways To understand the mechanisms by which UCN 01 induces more information G2M phase arrest, we studied intracellular signalling through western blot analyses. UCN 01 has been reported to inhibit chk1 and abrogate the G2M checkpoint in human colon carcinoma HCT116 cells Inhibitors,Modulators,Libraries and to induce G1 arrest in other human cancer cells. however, there are no reports on the effects of UCN 01 on hepatoma cell lines. Furthermore, we investigated UCN 01 induced G2M phase arrest, which has not been studied previously. Thus, our work investi gates the mechanism of UCN 01 induced G2M phase arrest in Huh7, HepG2, and Hep3B cell lines.
There are two key pathways involved in normal mammalian cell cycle arrest p53p21waf1 and CHK2CDC25C. Figure 3A indicates that p21Waf1 expression increased in a UCN 01 dose dependent manner in HepG2 cells Inhibitors,Modulators,Libraries but not in Huh7 or Hep3B cells. Increased p53 phosphorylation was observed in Huh7 and HepG2 cells, even at very low concentrations of UCN 01. However, total cellular p53 protein levels were not increased even up to 300 nM UCN Inhibitors,Modulators,Libraries 01 in any of the cell lines. Figure 3B indi cates that CHK2 phosphorylation increased in a UCN 01 dose dependent manner in all three cell lines, while CDC25C protein levels decreased in all cell lines. We also examined cyclin B levels in each cell line. The Cdc2cyclin B complex is the key enzyme regulating the G2 to M transition and is controlled by phosphorylation at various sites.
Inhibitors,Modulators,Libraries Therefore, we monitored cyclin B levels in each cell line after treatment with UCN 01. The results show that cyclin B levels decreased in a dose dependent manner in all three cell lines. Cell invasion is inhibited by UCN 01 Hepatoma cell invasion was assayed using the Matrigel invasion chamber. After 72 h, the lower layer mem branes were fixed and stained, and the number of cells in vading through the membrane was counted. Huh7 cells exposed to UCN 01 showed a significant de crease in the number of cells able to invade across Matrigel coated membranes compared with untreated cells. In contrast, UCN 01 treatment Inhibitors,Modulators,Libraries of HepG2 cells and Hep3B cells did not affect their invasive ability. We compared the proliferation rates of Huh7 cells cultured in 10% FBS or in serum free media.
There were no signifi cant differences between the total number of Huh7 cells in serum free media treated with various UCN 01 concen trations. This result demonstrates that UCN 01 mediated invasion inhibition is inhibitor expert not due to inhibition of proliferation. For the 30, 100, and 300 nM UCN 01 treated groups, the percentages of Huh7 cells in vading through the membrane were 32. 29%, 12. 29%, and 0%, respectively.