| 摘要: | 絞股藍皂苷是絞股藍萃取物中重要的組成，且有多種不同的藥理性，包括有抗腫瘤的增生及腫瘤的形成。最近研究顯示，在許多人類的癌細胞株中，絞股藍皂苷能誘發細胞週期停滯及引發細胞凋亡。然而在人類口腔癌細胞株的研究中，並沒有關於絞股藍皂苷對於DNA損傷，DNA修復的相關基因表現，細胞凋亡及預防細胞侵襲的相關報導。
本實驗的目的，為研究絞股藍皂苷對於人類口腔癌細胞株SAS其DNA損傷的影響，DNA修復的相關基因表現，細胞凋亡及細胞移動或侵襲的影響。
流式細胞儀被使用來檢測及量化SAS細胞的存活率，彗星試驗的結果顯示在絞股藍皂苷不同濃度的作用下，DNA損傷(彗星拖尾)的程度有濃度的依賴性。又在Real-time PCR的檢測下，當絞股藍皂苷的濃度為180 μg/ml，作用於SAS細胞株24小時後，將使14-3-3σ、DNA-PK、p53、ATM、ATR、BRCA1 的mRNA表現量減少。
流式細胞儀分析結果也顯示絞股藍皂苷誘導SAS細胞株細胞週期停滯在S期，且在DAPI實驗及DNA凝膠電泳的檢測下證明細胞凋亡的存在。又絞股藍皂苷誘導細胞中 ROS 、 Ca2+、 ER stress的表現增加，同時降低粒腺體膜電位的表現，引發Cytochrome c 的釋放。再由西方墨點法分析結果顯示絞股藍皂苷經由減少Bcl-2的表現，增強 Bax的表現量，進而降低粒腺體膜電位，造成Cytochrome c 的釋放，接著活化下游 Caspase-9 ,-3的活性，或藉由誘導 Endo G 從粒腺體的釋放，而導致細胞凋亡。
除了PI3K之外，絞股藍皂苷在降低SOS、Ras、uPA、FAK、Akt 、NF-κB、COX-2、 ERK1/2 、 MMP-9、 MMP-2的蛋白表現量方面，有時間的依賴性。另外絞股藍皂苷能減少MMP-2, MMP-7, MMP-9的 mRNA表現，但在FAK與Rho 其mRNA並無明顯變化。
綜合上述數據顯示， 絞股藍皂苷對人類口腔癌細胞株SAS能造成 DNA 損傷，使細胞週期停滯，同時抑制DNA 的修復，誘導細胞凋亡，並且抑制細胞的轉移和細胞侵襲的能力。
Gypenosides (Gyp) is the major component of Gynostemma pentaphyllum Makino extracts and is known to have diverse pharmacologic activities including anti-proliferation and anti-cancer effects. Recently, Gyp has been shown to induce cell cycle arrest and apoptosis in many human cancer cell lines. However, there is no available information to address the effects of Gyp on DNA damage, DNA repair associated gene expression, apoptosis and in preventing invasion in human oral cancer cells.
The purpose of this study was to investigate the effects of Gyp treatment on DNA damage, DNA repair gene expression, apoptosis and the migration and invasion in human oral cancer SAS cells.
Flow cytometry analysis was used to examine and quantitate the percentage of viable SAS cells. The results from Comet assay indicated that the incubation of SAS cells with various doses of Gyp led to a longer DNA migration smear (Comet tail) when compared with control and these effects are in a dose-dependent manner. The results from real time PCR analysis indicated that 180 μg/ml of Gyp for 24 h treatment in SAS cells led to decrease 14-3-3 proteins sigma (14-3-3σ), DNA-dependent serine/threonine protein kinase (DNA-PK), p53, ataxia telangiectasia mutated (ATM), ataxia-telangiectasia and Rad3-related (ATR) and breast cancer gene 1 (BRCA1) mRNA expression.
Flow cytometry analysis also indicated that Gyp induced cell cycle arrest (S phase arrest) and apoptosis in SAS cells.
Gyp induced production of ROS and Ca2+ and ER stress and decreased the levels of ΔΨm, causing Cytochrome c release. Western blotting results demonstrated that Gyp decreased Bcl-2 and increased Bax levels, decreased the levels of ΔΨm, caused Cytochrome c release, activations of Caspase-9 and -3, or induced Endo G release from mitochondria leading to apoptosis.
Gyp decreased SOS, Ras, urokinase-type plasminogen activator (uPA), focal adhesion kinase (FAK), RAC-alpha serine/threonine-protein kinase (Akt) , nuclear factor kappa B (NF-κB), cyclooxygenase-2 (COX-2), extracellular signal-regulated kinase 1/2 (ERK1/2) and matrix metalloproteinase -9, -2 (MMP-9, MMP-2) in a time-dependent manner except phosphatidylinositol 3-kinase (PI3K) levels. In addition, Gyp decreased the mRNA levels of MMP-2, MMP-7 and MMP-9 but not FAK and Rho in SAS cells.
These observations may offer information for the molecular mechanisms leading to cell apoptosis by Gyp in SAS cells. Taken together, Gyp induced DNA damage, inhibited the DNA repair, arrested the cell cycle, induced apoptosis and inhibited the metastatic and invasive capacity of oral cancer cells in vitro. |
