PA-22引起人類子宮頸癌細胞G2/M期停滯與細胞凋亡; PA-22 induced G2/M arrest and apoptosis in human cervical HeLa cells

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Title:	PA-22引起人類子宮頸癌細胞G2/M期停滯與細胞凋亡;PA-22 induced G2/M arrest and apoptosis in human cervical HeLa cells
Authors:	許日昇;Jih-Sheng Hsu
Contributors:	中國醫藥大學：醫學研究所碩士班
Keywords:	苦蘵;子宮頸癌;細胞凋亡;細胞週期;Physalis angulata;HeLa cells;apoptosis;cell cycle;G2/M arrest
Date:	2007-06-12
Issue Date:	2009-08-13 14:50:29 (UTC+8)
Abstract:	研究目的：本研究為首先探討PA-22對於人類子宮頸癌症的抗癌效果。研究方法：第一階段透過分子細胞的模式進行實驗，培養人類子宮頸癌細胞株，給予PA-22不同濃度及時間，以MTT細胞活性試驗、流式細胞儀、DAPI 細胞螢光染色法、DNA 階梯狀斷裂電泳法、Comet assay 單細胞凝膠電泳法、二維電泳法、西方點墨法。第二階段進行動物實驗，以兩週大的雌性嚴重免疫缺乏鼠 (Scid mice)，皮下植入人類子宮頸癌細胞株4x106，待生長至200mm3時，開始給予不同濃度PA-22，觀察體重與腫瘤大小變化。研究結果：PA-22有效的抑制細胞生長，是透過引起癌症細胞週期停滯在G2/M階段，與產生細胞凋亡。細胞週期的停滯是透過二種方式，影響相關蛋白表現；增加p21WAF1/Cip、 p27Kip1、p53、Wee1和降低Cyclin B1、Cdc2、Cdc25C、α-tubulin和 β-tubulin。PA-22也增加不具活性的磷酸化Cdc2 和Cdc25C。而PA-22引起的週期停滯和細胞凋亡，可以被p53 抑制劑~pifithrin α所阻斷；證明PA-22所產生的週期停滯作用，是經由依靠p53路徑和非依靠p53路徑。 PA-22所引發的細胞凋亡，是經由影響粒線體；透過改變Bax/ Bcl-2 比率，導致粒線體膜電位損失，cytochrome c 釋放，以及活化的caspase-8，caspase-9，與caspase-3。我們發現，PA-22也增加HSP70-2，HSP-90，以及Nucleophosmin的濃度。證明PA-22所產生的細胞凋亡作用，可能是經由粒線體路徑和非粒線體路徑。更進一步，藉由嚴重免疫缺乏品系之小鼠實驗，明顯的證明，PA-22可以抑制植入皮下的人類子宮頸癌細胞生長。研究結論：綜合以上實驗結果，PA-22透過調控p53蛋白，引起人類子宮頸癌細胞週期停滯和細胞凋亡。 Purpose : This study is the first investigated in regarding to the anticancer effect of PA-22 on human cervical HeLa cancer cells. Experimental Design : In vitro : PA-22 treated HeLa cells dose and time-dependently by MTT assay, Flow cytometry, DAPI assay, DNA Ladder assay, Comet assay, 2-D gel and Western blot. In vivo : Female Scid mice, 2 weeks of age, were purchased from National Laboratory Animal Center (Taipei, Taiwan). Mice were injected subcutaneously with 4x106 HeLa cells in 0.2 ml. After 2 weeks, when established tumors of ~200 mm3 were detected, treated PA-22 with different concentration. Observed changes of body weight and tumer size. Result : PA-22 has exhibited effective cells growth inhibition by inducing cancer cells to undergo G2/M phase arrest and apoptosis. Blockade of cells cycle was associated with increased levels of p21WAF1/Cip1, p27K ip1, p53, Wee1 and reduced amounts of Cyclin B1, Cdc2, Cdc25C in examined HeLa cells, α-tubulin and β-tubulin. PA-22 treatment also enhanced the levels of inactivated phosphorylated Cdc2 and Cdc25C. Blockade of p53 activity by pifithrin-α (a p53 inhibitor) partially decreased PA-22-induced G2/M arrest and apoptosis, suggesting it might be operated by p53-dependent and independent pathway. PA-22 treatment triggered the mitochondrial apoptotic pathway was indicated by a change in Bax/Bcl-2 ratios, resulting in mitochondrial membrane potential loss, cytochrome c release, and active caspase-8, caspase-9, and caspase-3. We also found out that PA-22 increased levels of HSP70-2, HSP-90, and nucleophosmin. Based on those observations It suggesting PA-22 might be operated by mitochrondia-dependent and independent apoptosis pathway. Further investigation revealed that PA-22 induced the inhibition of HeLa cells growth effect was also evident in a scid mice model. Conclusion : Taken together, these results suggest a critical role for p53 in PA-22-induced G2/M arrest and apoptosis of human cervical HeLa cancer cells.
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