Our previous study identified both high p27kip1 and low CKS1B protein levels were independent prognosis markers in hepatocelular carcinomas (HCC), however, CKS1B overexpression implicated clinical aggressiveness of HCC but not p27Kip1 protein turnover. In this study, we demonstrated a cytotoxic compound, goniothalamin (GTN), that stabilized p27kip1 protein and downregulated CKS1B mRNA as well as protein levels in HCC-derived cell lines. The IC50 of TP53-negative Hep-3B and TP53-positive SK-Hep1 after treatment with GTN for 48 h were detected as 25 and 10 £gM, respectively. The GTN induced DNA damages, formation of £^H2AFX foci, and upregulation of £^H2AFX protein levels in dose-dependent manners in both cell lines. In addition, GTN arrested Hep-3B and SK-Hep1 cells in G2/M and G1 phases, respectively. Protein levels of several cell cycle regulators, including p27Kip1 in Hep-3B and, p21Cip1 and CKS1B in SK-Hep1 cells have been noticeable upregulated after treatment with GTN. In Hep-3B cells, GTN induced the configuration of p27Kip1/CCND1, p27Kip1/CDK2; p27Kip1/CCNB1 and p27Kip1/CDK1, however, repressed the p27Kip1/STMN1 complexes. On the other hand, GTN evidently induced the configuration of p21Cip1/CDK2 and p21Cip1/CCNE1, but repressed the p21Cip1/SKP2 and p21Cip1/CKS1B complexes, in SK-Hep1 cells. The CKS1B protein abundance that was induced by GTN was stemmed from the CKS1B transactivity. However, GTN-induced p27Kip protein profusion in Hep-3B cell line was, instead, due to the stabilization of p27Kip1 protein, mainly in the cytosol. Both GTN and the proteasome inhibitor, MG132, induced p27Kip1 ubiquitination and p27Kip1 protein profusion with an additive effect, regardless of CKS1B protein level, suggesting that the p27Kip1 protein might be regulated by GTN through an alternative ubiquitin-complex in Hep-3B cells. In addition to the quantitative reverse transcription -polymerase chain reaction, GTN-induced p21Cip1 protein level in SK-Hep1 cells could be traced back to mRNA level, by evidence of quantitative immunoprecipitation/chromatin immunoprecipitation assay with primer sets specific to two regions of the p21Cip1 proximal promoter. Taken together, GTN is able to induce the cell cycle arrest via stabilization of p27Kip1 protein and downregulation of CSK1B mRNA and subsequent protein levels in TP53-negative Hep-3B and TP53-positive SK-Hep1 cells, respectively. The GTN-repressed CKS1B transcription and thereafter, cell cycle arrest, was irrelevant to the p27Kip1 protein stability in Hep-3B cells.
| Identifer | oai:union.ndltd.org:NSYSU/oai:NSYSU:etd-0209110-160851 |
| Date | 09 February 2010 |
| Creators | Liu, Hui-wen |
| Contributors | Hurng-wern Huang, Pei-jung Lu, Long-sen Chang, Yow-ling shiue |
| Publisher | NSYSU |
| Source Sets | NSYSU Electronic Thesis and Dissertation Archive |
| Language | English |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | http://etd.lib.nsysu.edu.tw/ETD-db/ETD-search/view_etd?URN=etd-0209110-160851 |
| Rights | not_available, Copyright information available at source archive |
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