| 摘要: | 摘要 Norcantharidin (NCTD)是斑蝥素的合成衍生物，有抗癌的特性，現用於臨床上抗肝腫瘤。然而有關NCTD對人類肝癌細胞作用之研究文獻甚少。因此本論文將以人類肝癌細胞株為研究模式探討NCTD在癌細胞內之分子作用機制，將HepG2, Hep3B 與 Huh-7等肝癌細胞株經由NCTD處理48小時後，NCTD抑制細胞增生，造成細胞生長停滯於有絲分裂期，而後引起細胞凋亡。經由5 μg/ml的NCTD處理後，Cdc25C 與 p21CIP1/WAF1蛋白質的表現增加且會被磷酸化。此外，NCTD處理48小時後造成cyclin B1-associated histone H1 kinase的活性增加，但在72小時後，cyclin B1蛋白質的表現量減少將近70%，同時cyclin B1脢活化之現象也減少。經由NCTD處理者明顯的p53蛋白會減少，但Cdk1 與 p27KIP1的表現不會改變。另外，經由NCTD處理之細包內之Bcl-2 與 Bcl-XL被磷酸化，但細胞中之Bax 或Bad的表現並無變化。NCTD處理的HepG2細胞株中，以免疫複體偵測到與Bcl-2結合的Bax減少。所以Bcl-2之磷酸化似乎抑制了與Bax之結合能力。同時經由NCTD處理之細胞caspase-9 與caspase-3被活化，接著進行DNA斷裂與細胞凋亡的細胞特徵。如預先處理廣效的Caspase抑制劑z-VAD-fmk則明顯的抑制NCTD造成的caspase-3活化與細胞的死亡。由本論文之實驗結果可推論磷酸化P21CIP1/WAF1 與 Cdc25C 會雙向調節cyclin B1-associated kinase 的活性，是NCTD造成細胞週期M phase的停留的主要作用分子。同時，增加p21CIP1/WAF1的表現及Bcl-2 與Bcl-XL的磷酸化，caspase-9 與caspase-3的活化，為NTCD造成細胞凋亡的重要分子機轉。; Abstract Norcantharidin (NCTD) is a demethylated form of cantharidin with antitumor properties, which is now in use as a routine anticancer drug against hepatoma. However, there is limited information on the effect of NCTD on human cancer cells. In the present study, NCTD inhibited proliferation, caused mitotic arrest, then progressed to apoptosis within 96 h in three human hepatoma cell lines: HepG2, Hep3B and Huh-7. NCTD treatment (5 μg/ml) enhanced the expression of Cdc25C and p21CIP1/WAF1, increasing the phosphorylation of these two proteins. In addition, NCTD treatment induced an earlier increase in cyclin B1-associated histone H1 kinase activity within 48 h, but an approximately 70% reduction of both protein level and kinase activity of cyclin B1 was observed at 72 h. Treatment with NCTD significantly decreased the expression of p53 protein, but did not affect the expression of Cdk1 and p27KIP1. Moreover, NCTD treatment also increased the phosphorylation of Bcl-2 and Bcl-XL, but did not affect the expression of Bax or Bad. Bcl-2 phosphorylation seems to inhibit its binding to Bax was detected in immunocomplex with Bcl-2 in NCTD-treated HepG2 cells. In addition, NCTD treatment caused the activation of caspase-9 and caspase-3, preceding DNA fragmentation and morphologic features of apoptosis. Preteament with broad-spectrum caspase inhibitor z-VAD-fmk markedly inhibited NCTD-induced caspase-3 activity and cell death. These results suggest that phosphorylation of P21CIP1/WAF1 and Cdc25C and biphasic regulation of cyclin B1-associated kinase activity may contribute to the NCTD-induced M phase cell cycle arrest. Furthermore, the increase of p21CIP1/WAF1, phosphorylation of Bcl-2 and Bcl-XL, activation of caspase-9 and caspase-3 may be the molecular mechanism through which NCTD induces apoptosis. |
