Chamaecyparis obtusa var. formosana 扁柏精油誘導A549人類肺癌細胞凋亡與抑制細胞轉移之研究

Effect of Essential Oil from Chamaecyparis obtusa var. formosana Heartwood on Apoptosis and Inhibitory Migration of A549 Human Lung Cancer Cell Line.

陳靖宜(Jing-Yi Chen)

指導教授 ： 胡超群

中山醫學大學/健康管理學院/應用化學系碩士班/碩士(2011年)

https://doi.org/10.6834/CSMU.2011.00118

摘要

台灣扁柏 ( COF ) 是一種多年生本土植物，分佈於台灣中北部山區。市售之扁柏精油大都經由水蒸氣蒸餾其心材部分離製造。本研究首先以 MTT assay 評估含台灣扁柏內共九種市售精油對三種人類腫瘤細胞株 SK-Hep-1 、 HepG2 ( 肝癌 ) ， DLD1 ( 大腸癌 ) 之細胞毒殺能力，發現扁柏精油 ( IC50 93.1 至 243.2 μg/ml ) 較其他精油 ( IC50 69.4 至 1461.7 μg/ml ) 對三種癌細胞均有較佳抑制生長能力，基於肺癌為癌症中最常見者，本研究選取人類肺腺癌細胞株 A549 探討扁柏精油對其生化活性，發現 50 ~ 150 μg/ml 之濃度下即能明顯抑制 A549 細胞生長，並誘發凋亡反應。流式細胞儀測定結果顯示，扁柏精油促使 A549 細胞停滯在 S 期及 G2/M 期，西方墨點法顯示與細胞週期相關之蛋白質 cyclin A 、 B1 、 E 及 cyclin-dependent kinases CDK2 、 CDK4 均有下降之趨勢，扁柏精油同時可降低調控蛋白 Bax/Bcl-2 和 Bax/Bcl-xL 之比率有利凋亡機轉之進行，同時亦觀察到細胞凋亡下游蛋白 caspase-3/-7/-9 活化型與 PARP 蛋白降解有增加之趨勢。本研究進一步由 wound healing 與 Boyden chamber 結果發現精油亦有抑制癌細胞轉移及侵入之作用。此外，扁柏精油可有效地抑制 Akt 蛋白磷酸化之表現，減少 MMP-1、MMP-2 及uPA之表現，另外亦可抑制 NF-κB 抑制蛋白 IκB 之磷酸化，導致 NF-κB 在細胞質之累積，進而調控 MMP-1、MMP-2 及uPA 之癌細胞轉移相關蛋白之表現，扁柏精油除了以上作用外，亦具有抑制發炎反應中 NO 合成酵素 iNOS 及 cyclooxygenase 之作用，綜合研究結果，扁柏精油無論在癌症治療及預後均可視為有效之醫療資源。

關鍵字

台灣扁柏 ； 精油 ； 肺癌 ； 細胞週期 ； 細胞凋亡 ； 腫瘤轉移

並列摘要

Chamaecyparis obtusa var. formosana ( COF ) is native species distributed at the northern and central mountains of Taiwan. The essential oil of COF is complex mixtures of volatile and odorous compounds derived from secondary tree metabolism. It can be extracted from heartwood by hydro- and steam-distillation and is known to induce a wide range of biological effects through their antibacterial, antifungal, antioxidant activities. In order to explore its beneficial properties on human cancer cells, we investigated and compared the cytotoxic effects of COFH from eight other essential oils to human carcinoma cell line HepG2, SK-Hep1, and DLD1. In this study, essential oil of COFH was more effective against tested cell lines ( IC50 93.1 to 243.2 μg/ml ) than that from eight other essential oils ( IC50 69.4 to 1461.7 μg/ml ). As lung cancer is the mostly suffered cancer type in the world. In this study, we demonstrated that this essential oil was also shown to inhibit proliferation and induce apoptosis under the concentration from 50 to 150 μg/ml in the human lung adenocarcinoma cell line: A549. The cell cycle of these extract-treated cells was arrested at the S to G2/M phase as determined by flow cytometry. Western blotting analysis revealed that COFH markedly decreased the expression of cyclins ( A1, B and E ) and cyclin-dependent kinases ( Cdk2 and Cdk4 ). COFH treatment led to augmentation of Bax/Bcl-2 and Bax/Bcl-xL ratios to favor apoptosis in A549 cancer cells. However, the proteolytic cleavage of poly ADP-ribose polymerase ( PARP ) and the activation of pro-apoptotic caspase-3/-7/-9 were all increased upon treatments of this oil. This study also investigates the anti-metastatic effect of COFH in A549 cell. Our study first noted that COFH inhibited A549 cells invasion and migration by wound-healing assay and Boyden chamber assay. The data also showed that COFH could inhibit phosphorylation of Akt and extracellular signal-regulated kinase 1 and 2 ( ERK1/2 ), decrease the protein levels of matrix metalloproteinase-1 ( MMP-1 ), matrix metalloproteinase-2 ( MMP-2 ) or urokinase-type plasminogen activator (u-PA). Next, COFH also strongly inhibited the phosphorylation of inhibitor of kappa B alpha ( IκB-α ) causing NF-κB stabilization and thus enhanced cytoplasmic accumulation of NF-κB,which is involved in the up-regulating MMP-1, MMP-2 or u-PA. The essential oil also block expression of NO synthase ( iNOS ) and cyclooxygenase-2 ( COX-2 ) in the LPS-stimulated cells. Our result suggests that the essential oil of COFH could be a potential medicinal resource to cancer therapy and prognosis.

並列關鍵字

Chamaecyparis obtusa var ； formosana ； essential oil ； lung cancer ； cell cycle ； apoptosis ； tumor metastasis

參考文獻

簡世昌 ，台灣杉皮部與台灣扁柏心材成分之研究。臺灣大學化學所博士論文， 2004 。(台灣大學郭悅雄教授實驗室)

Bassi R, Heads R, Marber MS, Clark JE. 2008. Targeting p38‐ MAPK in the ischaemic heart: kill or cure? Curr Opin Pharmacol 8: 141–146.

Basseres DS, Baldwin AS. 2006. Nuclear factor-kappaB and inhibitor of kappaB kinase pathways in oncogenic initiation and progression. Oncogene 25: 6817–6830.

Braun-Dullaeus RC, Mann MJ, Dzau VJ. 1998. Cell cycle progression: new therapeutic target for vascular proliferative disease. Circulation 98: 82–89.

Calcabrini A, Stringaro A, Toccacieli L et al. 2004. Terpinen-4-ol, the main component of Melaleuca alternifolia (tea tree) oil inhibits the in vitro growth of human melanoma cells. J Invest Dermatol 122: 349–360.