Squamocin引起人類肝癌細胞HepG2細胞週期停滯與凋亡之機制探討

Squamocin causes cell cycle arrest and apoptosis in human hepatocellular carcinoma HepG2 cells

黃宇君(Yu-Chun Hwang)

指導教授 ： 吳志中

高雄醫學大學/藥學院/天然藥物研究所碩士班/碩士(2009年)

摘要

在台灣，惡性腫瘤已變成致死疾病的第一名。根據衛生署統計，肝癌是第二常見的腫瘤疾病。除了肝癌細胞對於藥物的抗藥性高，病患的肝功能損壞也讓藥物的使用上得審慎評估。目前臨床上使用的化療藥品與分子標的藥物為主的全身療法仍無法有效治療肝癌。因此研發新的抗肝癌藥物更是刻不容緩的任務。
天然物一直被視為是新藥研發的寶庫。本實驗室從番荔枝科植物中分離出acetogenin類的化合物squamocin。先前的研究已經發現squamocin具有抗癌效果。在本實驗中使用HepG2人類肝癌細胞進行實驗，發現squamocin在極低的濃度(0.5~5nM)就可以抑制HepG2生長。利用流式細胞儀分析細胞週期，發現squamocin可以造成HepG2的細胞週期停滯於G1與G2/M期。Squamocin使cyclin D、 Cdk2與Cdk4等調控G1期量的蛋白質減少。此外squamocin也可導致hypophosphorylated pRB的增加與E2F1 表現量下降。在G2期相關蛋白質方面，squamocin可引起Cdk1與Cdc25C的減少，並可造成Cdc25C的磷酸化。進一步探討Cdc25C上游的調控者，我們發現squamocin會引起Chk1、Chk2與p38的活化。這表示Chk1、Chk2與p38在squamocin引起的Cdc25C抑制現象與後續產生的細胞週期停滯可能扮演重要角色。最後，我們觀察到高濃度的squamocin （5nM）在長時間作用(48h)下會造成HepG2的細胞凋亡。進一步檢測caspase凋亡路徑相關蛋白質，並使用流式細胞儀分析caspase抑制劑- ZVAD-fmk對squamocin的影響，發現squamocin使HepG2細胞產生caspase非依賴性的細胞凋亡。
本研究發現HepG2對squamocin的高敏感，使squamocin可能具有潛力成為新的抗肝癌藥物。尤其它對於HepG2確實的分子標的值得我們進一步探討。

關鍵字

番荔枝科 ； 細胞週期停滯 ； 細胞凋亡

並列摘要

Malignant neoplasm has become the leading cause of death in Taiwan. According to stastistic data from Department of Health, hepatocellular carcinoma (HCC) is the second common cancer in Taiwan. At present, systemic therapies including chemotherapy and molecular targeted therapy can not effectively treat this malignancy because of resistance to drugs and descent of tolerance caused by liver dysfunction. Therefore, it is an emergent task to find new drug candidates for treating HCC.

Natural products represent a rich source of new chemical compounds for developing anticancer drugs. In previous studies, squamocin, an Annonaceous acetogenin, has been reported as a potential anticancer agent. In the present study, we found that squamocin inhibited the growth of HepG2 cells at very low concentrations (0.5~5nM). Flow cytometry showed that squamocin induced cell cycle arrest at G1 and G2/M phase in HepG2 cells. Western blotting revealed that squamocin decreased the expression of cyclin D, Cdk2 and Cdk4. Besides, squamocin treatment also led to an increase in hypophosphorylated pRB and a decrease in E2F1 protein levels. In G2 phase-related proteins, squamoicin decrease Cdk1 and Cdc25C and induced Cdc25C Ser216 phosphorylation. To analysis the upstream regulators of Cdc25C, we observed the activation of Chk1, Chk2, and p38 after treatment of HepG2 cells with squamocin. The results suggested Chk1, Chk2 and p38 might be responsible for squamocin-induced Cdc25C downregulation and thus led to cell cycle arrest in G2/M phase. In addition, prolonged treatment (48h) of HepG2 cells with a high concentration of squamocin (5nM) induced apoptotic death. However, squamocin did not activate caspase cascade, suggesting that squamocin-induced apoptosis is caspase-independent.

In conclusion, based on its very potent and novel activity, squamocin can be considered as a promising anticancer drug, and it is worthy to find the special target of squamocin in HepG2 cells.

並列關鍵字

squamocin ； cell cycle arrest ； caspase-indepedent apoptosis

參考文獻

參考文獻

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