The goniothalamin (GTN) and related styryl-lactones were found to be cytotoxic and apoptotic
to a variety of tumor cell lines including breast cancer MCF-7, cervical cancer HeLa and leukemia
HL60. In HL60 cells, GTN triggers mitochondria dysfunction, caspase activation and eventually,
apoptosis. In this study, we demonstrated that GTN was able to induce apoptosis in hepatocellular
carcinoma derived cells, SK-Hep1 and Hep-3B cells via upregulation of the phorbol-12-myristate-13-induced 1 (PMAIP1) protein , alternation of mitochondrial membrane potentials (P < 0.05) via TP53-dependent and -independent transactivations. Treatment with GTN induced DNA damage, formation of reactive oxygen species (ROS, P < 0.05) and activated cleaved CASP8, CASP9, CASP3 and poly (ADP-ribose) polymerase 1 proteins. However, cleaved BH3 interacting domain (BID) death agonist protein was not identified, suggesting that an intrinsic cellular stress was produced after GTN treatments in both cell lines. A pan caspase inhibitor, z-VAD-FMK, suppressed GTN-induced apoptotic cell percentages (P < 0.05), demonstrating that GTN-induced apoptosis was mediated by the activation of the caspase cascade protein. The GTN induced ROS formation prior to DNA damage in SK-Hep1, yet in reverse order in Hep-3B cells.
Moreover, GTN induced DNA damages through activation of the gamma H2A histone family
member X (£^H2AFX, Ser139)/phospho-CHK1 checkpoint homolog (pCHEK1, Ser345)/phospho-CHK2 checkpoint homolog (pCHEK2, Thr68)/phosphos-tumor protein p53 (pTP53, Ser15), and £^H2AFX/pCHEK2 (Thr68), in SK-Hep1 and Hep-3B cells,respectively.
Among five integral outer mitochondrial membrane proteins that blocks or induces apoptotic death,
PMAIP1 protein and PMAIP1 mRNA levels were upregulated after GTN treatments for 4 to 6 h in
both cell lines (P < 0.05). Transfection of shRNA interference plasmids targeting PMAIP1 gene
downregulated PMAIP1 mRNA (P < 0.05) and PMAIP1 protein (P < 0.05) levels, as well as GTN-induced apoptotic cell percentages (P < 0.05) in both cell lines. In parallel, transfection of the
shRNA interference plasmid targeting TP53 gene in SK-Hep1 cells, downregulated TP53 and PMAIP1 mRNA (P < 0.05) and TP53, pTP53, PMAIP1, cleaved PARP1 protein levels and apoptotic cell percentages (P < 0.05). Treatment with the TP53 inhibitor, PFT-£\ or transfection of a dominant negative TP53 plasmid, pTP53mt135, repressed TP53, pTP53 and PMAIP1 protein and/or TP53, PMAIP1 mRNA levels (P < 0.05), however, significantly augmented GTN-induced apoptotic cell percentages (P < 0.05). Cytosol/mitochondria fractionation identified that TP53, along with PMAIP1 proteins, were translocated to mitochondria after GTN treatment in time-dependent manners. Taken together, our studies demonstrated that GTN induced apoptosis via PMAIP1 via both TP53-dependent and -independent transactivation pathways. In TP53-positive SK-Hep1 cells, PMAIP1 and TP53 proteins conducted synergic effects.
| Identifer | oai:union.ndltd.org:NSYSU/oai:NSYSU:etd-0209110-094932 |
| Date | 09 February 2010 |
| Creators | Huang, Yu-ting |
| Contributors | Long-sen Chang, Yow-ling shiue, Pei-jung Lu, Hurng-wern Huang |
| 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-0209110-094932 |
| Rights | not_available, Copyright information available at source archive |
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