合成的苯乙酸衍生物對人類鱗狀上皮肺癌細胞CH27之生長抑制作用; Growth Inhibitory Effects of Synthetic Phenylacetic acid Derivatives on Human Squamous Lung Cancer CH27 Cells

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Title:	合成的苯乙酸衍生物對人類鱗狀上皮肺癌細胞CH27之生長抑制作用;Growth Inhibitory Effects of Synthetic Phenylacetic acid Derivatives on Human Squamous Lung Cancer CH27 Cells
Authors:	詹淑秦;Hsu-Chin Chan
Contributors:	中國醫藥學院藥物化學研究所
Keywords:	苯乙酸衍生物;細胞凋亡;細胞週期;生長抑制;phenylacetic acid derivatives;apoptosis;cell cycle;growth inhibitory
Date:	1992
Issue Date:	2009-12-23
Abstract:	苯乙酸是一毒性很低的分化誘導劑且有抗癌活性，本論文研究是將一系列的苯乙酸衍生物對人類鱗狀上皮肺癌細胞CH27進行活性篩選，希望能找出更有效的苯乙酸類似物。結果顯示這一系列的化合物都有明顯的抗癌細胞增殖活性，而其中有兩個化合物其作用活性比苯乙酸強，它們是4-fluoro-N-butyl phenylacetamide (H6)與2-fluoro-N- butyl-phenylacetamide (SCK6) ，為了探討H6 與 SCK6的抗腫瘤細胞增殖的機轉，於是採用螢光染色法與DNA膠體電泳分析，結果顯示H6 與 SCK6能誘導人類鱗狀上皮肺癌細胞CH27細胞進行凋亡，而兩者所引起的細胞凋亡主要歸因於細胞質內cytochrome c的累積與caspase cascade的活化，特別是caspase-9與caspase-3。 H6因誘導caspase的活化，引發poly (ADP-ribose) polymerase蛋白分解斷裂，接著造成DNA無法修補而斷成特殊的片段，如以caspase抑制劑前處理，則能明顯的抑制H6所誘導的caspase活性與細胞凋亡，除此之外，H6誘導細胞凋亡伴隨著Bcl-Xs蛋白質的表現增加，這些結果顯示，H6造成細胞凋亡可能是透過與Bcl-Xs蛋白質和caspase相關的機制。在SCK6抗增殖作用機制的研究上，我們先分析SCK6對人類鱗狀上皮肺癌細胞CH27細胞週期的影響，結果發現以1 mM SCK6處理會造成肺癌細胞停在細胞週期的G1期，分析一些與細胞週期相關的蛋白質變化，顯示SCK6處理後會使cyclin-dependent kinase 2(Cdk2), Cdk4, cyclin E 與cyclin D3蛋白質表現量降低，卻不會影響到Cdk6, cyclin D1 與 D2，另外SCK6的處理會造成p53 與 p21CIP1/WAF1蛋白質表現程度上升。而且SCK6誘導細胞凋亡伴隨著Bcl-2蛋白質的表現減少，我們利用腺病毒 - Bcl-2 - 載體感染肺癌細胞株，讓Bcl-2蛋白質在肺癌細胞內過量表現，再以SCK6處理能明顯的抑制SCK6誘導的細胞凋亡。若以caspase抑制劑前處理，同樣的能明顯抑制SCK6所誘導的細胞凋亡。歸納其總反應機制，我們認為SCK6是透過將G1期相關的細胞週期分子Cdks 和 cyclins負向調節以及p53 與 p21CIP1/WAF1蛋白質表現程度上升，而造成肺癌細胞週期停滯在G1期。除此之外，Bcl-2蛋白質表現的降低與caspase-9/caspase-3路徑的活化，可能是SCK6誘導細胞凋亡的機轉之一。; Phenylacetic acid is a differentiation inducer, has anticancer activity with relatively low toxicity. In the present study, we examined the anticancer effect of series synthetic phenylacetic acid derivatives in human lung cancer cells for searching more effective phenylacetic acid analogous. Results shown that the antiproliferative effects of these synthetic compounds were stronger than those of phenylacetic acid, and that 4-fluoro-N-butyl phenylacetamide (H6) and 2-fluoro-N-butyl-phenyl- acetamide (SCK6) are the most potent compounds. To address the mechanism of anti-proliferative effect of H6 and SCK6, DAPI staining, in situ TUNEL assay and DNA gel electrophoresis analysis indicated that a marked reduction in the number of CH27 cells with H6 and SCK6 were related to the induction of apoptosis. The apoptosis triggered by H6 and SCK6 was accompanied by the accumulation of cytosolic cytochrome c and activation of caspase cascade (caspase-9 and -3). H6 induce proteolytic cleavage of poly (ADP-ribose) polymerase, which followed the appearance of caspase activity and preceded DNA fragmentation. Pretreatment with caspase inhibitors markedly inhibited H6-induced caspase activity and apoptosis. This H6-induced apoptosis associated with upregulation of Bcl-Xs protein. These results suggest that H6 may induce apoptosis through a Bcl-Xs and caspase-dependent mechanism. To search the mechanism of antiproliferative effect of SCK6, cell cycle analysis was performed. Result showed that SCK6 induced G1 arrest in CH27 cells following 1 mM of drug exposure. Western blot analysis of G1 phase regulatory proteins demonstrated that the protein levels of cyclin-dependent kinase 2 (Cdk2), Cdk4, cyclin E and cyclin D3 were decreased after treatment with SCK6 but not Cdk6, cyclin D1 and D2. In contrast, SCK6 increased the protein levels of the p53 and p21CIP1/WAF1. This SCK6-induced apoptosis was accompanied by downregulation of Bcl-2 protein. Overexpression of Bcl-2 by adeno-Bcl-2 vector infection significantly inhibited SCK6-induced apoptosis. Moreover, treatment with caspase inhibitors also markedly reduced cell death induced by SCK6. Taken together, these results suggest that down-regulation of G1 -associated Cdks and cyclins and upregulation of p53 and p21CIP1/WAF1 may contribute to SCK6-mediated G1-phase arrest. Furthermore, the decrease of Bcl-2 and activation of caspase-9/caspase-3 may be the effector mechanism through which SCK6 induces apoptosis.
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