Aqueous Ocimum gratissimum extract inhibits cell growth of human hepatocellular carcinoma via induction of cell cycle arrest

由於肝臟是會再生的器官而使得肝癌患者有極高的復發率，現行的治療方式無法達到有效的治療效果，因此開發出一種無副作用的新藥物是迫切需要的。七層塔 (Ocimum gratissimum Linn) 屬於羅勒的一種，其功效為抗氧化、抗癌、抗肝臟纖維化等具有極高之藥用價值。本研究探討七層塔水萃物對於人類肝癌細胞株Hep3B、Hep3BX、SK-Hep1及HA22T生長之影響。結果發現七層塔水萃物能有顯著降低Hep3BX、SK-Hep1及HA22T細胞存活率且有濃度依賴的現象 (濃度0-800 μg/mL)，然而對於Hep3B細胞的影響卻不大。進一步以西方墨點分析試驗發現，七層塔抑制Hep3BX、SK-Hep1及HA22T的G1控制蛋白Cyclin-dependent kinase 4及Cyclin-dependent kinase 2的表現量也有濃度依賴的現象，確認細胞生長抑制現象存在。在細胞能量代謝的試驗中，人類肝癌細胞的氧氣消耗率與產酸率皆隨著處理濃度增加而受到抑制。因此這些研究結果推論，七層塔水萃物可能是通過抑制糖解作用 (glycolysis) 和粒線體功能 (mitochondrial function)，而導致肝癌細胞週期阻滯而抑制細胞生長。Hepatocellular carcinoma is a major health problem, and it is the second highest cause of cancer-related death in Taiwan, behind only lung. Five-year survival rates of up to 60 to 70% can be achieved in well-selected patients. However, disease that is diagnosed at an advanced stage or with progression after locoregional therapy has a dismal prognosis, owing to the underlying liver disease and lack of effective treatment options. Ocimum gratissimum Linn (OG) has been reported to show anti-oxidant, anti-carcinogenic and anti-fibrosis therapeutic effects. Here we aimed to investigate the effects of aqueous extract of OG leaf (OGE) on malignant liver cancer Hep3B, Hep3BX, SK-Hep1, and HA22T cell lines. Our results reveled that OGE decreased the cell viability of Hep3BX, SK-Hep1, and HA22T cells in a dose-dependent manner (range from 0-800 μg/mL), while there is little effect on Hep3B cells. Further investigation showed that the expressions of cell cycle checkpoint cyclin-dependent kinase 4 and cyclin-dependent kinase 2 were also decreased by treatment with OGE in Hep3BX, SK-Hep1, and HA22T cells in a dose-dependent manner suggesting cell growth arrest performance. Bioenergetic approach, using the Extracellular Flux Analyzer to analysis, revealed the OGE treatment led a dose-dependent decrease in oxygen consumption rate and extra cellular acidification rate in SK-Hep1 cells. Therefore, these findings suggest that OGE induced the cell cycle arrest may be mediated through inhibiting glycolysis and mitochondrial function in hepatocellular carcinoma cells.中英文摘要--------------------------------------------------------------------------1 縮寫表--------------------------------------------------------------------------------3 一、緒論-------------------------------------------------------------------------------4 二、研究動機與目的--------------------------------------------------------------11 三、實驗架構-----------------------------------------------------------------------12 四、實驗材料及方法--------------------------------------------------------------13 五、實驗結果-----------------------------------------------------------------------22 六、討論-----------------------------------------------------------------------------29 七、參考文獻-----------------------------------------------------------------------32 八、圖表與圖表說明--------------------------------------------------------------40 九、附錄-----------------------------------------------------------------------------5

Aqueous Ocimum gratissimum extract induces cell apoptosis in human hepatocellular carcinoma cells

Treatment of advanced hepatocellular carcinoma (HCC) has exhibited a poor overall survival rate of only six to ten months, and the urgency of the development of more effective novel agents is ever present. In this line of research, we aimed to investigate the effects and inhibitive mechanisms of aqueous Ocimum gratissimum leaf extract (OGE), the extract of Ocimum gratissimum, which is commonly used as a therapeutic herb for its numerous pharmacological properties, on malignant HCC cells. Our results showed that OGE decreased the cell viability of HCC SK-Hep1 and HA22T cells in a dose-dependent manner (from 400 to 800 µg/mL), while there is little effect on Chang liver cells. Moreover, cell-cycle analysis shows increased Sub-G1 cell count in SK-Hep1 and HA22T cells which is not observed in Chang liver cells. These findings raise suspicion that the OGE-induced cell death may be mediated through proteins that regulate cell cycle and apoptosis in SK-Hep1 and HA22T cells, and further experimentation revealed that OGE treatment resulted in a dose-dependent decrease in caspase 3 and PARP expressions and in CDK4and p-ERK1/2expressions. Moreover, animal tests also exhibited decreased HCC tumor growth by OGE treatment. We therefore suggest that the inhibition of cell viability and tumor growth induced by OGE may be correlated to the alteration of apoptosis-related proteins