探討紅球薑之萃取成份球薑酮抑制人類乳癌細胞轉移作用之相關路徑

Regulation of survival, proliferation, invasion, and metastasis of breast cancer cells possibly through modulation of MMPs and Hedgehog signaling pathways by the extract of Zingiber zerumbet — Zerumbone

陳盈羽(Ying-Yu Chen)

指導教授 ： 張菡馨

中山醫學大學/健康管理學院/營養學研究所/碩士(2011年)

https://doi.org/10.6834/CSMU.2011.00056

摘要

乳癌已躍居全世界及台灣女性癌症發生率之首。本研究以人類乳癌細胞株MDA-MB-468、MDA-MB-435S為實驗模式探討紅球薑萃取成份之球薑酮(Zerumbone)對乳癌細胞轉移作用(metastasis)之影響。本篇利用不同濃度之Zerumbone處理細胞株：MDA-MB-468為0、10、20μM，MDA-MB-435S則為0、10、50μM。進行細胞存活率、細胞基質侵襲能力試驗(Matrigel invasion assay)、移動試驗(Migration assay)。在高濃度下Zerumbone可顯著抑制MDA-MB-468侵襲率達到70.15% (P<0.01)，MDA-MB-435S更高達95% (P<0.05)；在移動試驗中， Zerumbone確實可顯著抑制細胞移動能力(P<0.05)。文獻指出發炎作用與癌症之形成有關，慢性且長期的發炎作用有助於癌化發生，以iNOS及COX2抗體偵測發炎作用之表現，結果發現在MDA-MB-468中以Zerumbone 20μM處理下，發炎蛋白表現有降低之現象，推測Zerumbone具有抗發炎之效用。為探討Zerumbone是否能抑制基質金屬蛋白酶(Matrix metalloproteinases)之活性進而抑制轉移。利用明膠蛋白酵素電泳法分析，結果發現，MDA-MB-435S培養液中的MMP-2活性顯著降低(P<0.05)；進一步分析細胞內蛋白質，發現兩細胞株於MMP-2活性表現呈現不同的趨勢：MDA-MB-468為8到16小時呈現遞減現象；MDA-MB-435S其16小時之表現量大於8小時，而MMP-2表現量與細胞移動試驗呈現正相關之趨勢。又由文獻得知Zerumbone與Hedgehog/GLI訊號傳遞路徑有關，利用西方墨點法偵測Hedgehog/GLI相關蛋白表現。結果發現，Zerumbone可誘發Hedgehog蛋白表現；Zerumbone處理細胞8、16小時後，發現tGLI1 (truncated GLI1)表現量增加，與細胞移動試驗趨勢相同；Zerumbone作用24小時後，GLI1表現減弱，活化細胞凋亡(apoptosis)相關蛋白質表現量，促進細胞死亡。本篇結果表示，Zerumbone可透過不同路徑達到抑制癌細胞之轉移作用。

關鍵字

球薑酮 ； 乳癌 ； 細胞凋亡 ； 轉移 ； 刺蝟基因

並列摘要

Breast cancer has ranked first among female cancers in Taiwan and the world. Base on the experiment model using breast cancer lines MDA-MB-468 and MDA-MB-435S, we investigated how the Zerumbone influences the metastasis of breast cancer cell lines. In this study, cell lines were treated with different concentrations of Zerumbone: for MDA-MB-468 were 0, 10, 20uM; as for MDA-MB-435S were 0, 10, 50uM, and we examined the viability of the cells, matrigel invasion assay and migration assay. High concentration of Zerumbone restrained the relative invasion rate (%) of MDA-MB-468, which has reduced 70.15% (P<0.01); moreover, the relative invasion rate of MDA-MB-435S dropped by 95% (P<0.05). Our results suggest Zerumbone effectively decreased the ability of migration of breast cancer cells. Past researches indicated that inflammatory was linked to carcinogenesis, and the chronic long-term inflammatory contributed to cancer. We used iNOS and COX2 antibodys to detect expression of inflammatory, and the result showed that the expression of inflammatory proteins had decreased in MDA-MB-468 with the treatment of Zerumbone 20uM. Therefore, we speculated Zerumbone could inhibit inflammatory. To investigate how Zerumbone inhibit the activity of matrix metalloproteinases and subsequently reduce metastasis, we applied gelatin zymography assay and found out the activity of MMP-2 in conditional medium of MDA-MB-435S decreased; furthermore, analyzing the cell protein we discovered the activity of the two cell lines in MMP-2 demonstrated different trends: from 8 to 16 hours, the MDA-MB-468 shows a descending phenomenon; as for MDA-MB-435S, after 16 hours, the activity of MMP-2 is higher than 8 hours. The relationship between expression of MMP-2 and metastasis was a positive correlation. The signal transduction pathway between Zerumbone and Hedgehog/GLI were related, and we used the Western Blot to examine how the related protein interacted. The result showed Zerumbone can induce the expression of Hedgehog. After 8 and 16 hours treating with Zerumbone, we found tGLI1 (truncated GLI1) expression increased, and strongly correlate with the results of migration. After 24 hours treatment, the expression of GLI1 decreased, while apoptosis increased, providing a possible cause of cell death. We conclude that Zerumbone is able to inhibit the metastasis of human breast cancer cells through different pathways.

並列關鍵字

Zerumbone ； breast cancer ； apoptosis ； metastasis ； hedgehog

參考文獻

Albini, A., & Sporn, M. B. (2007). The tumour microenvironment as a target for

Agalliu I, Gern R, Leanza S, Burk RD. Associations of highgrade prostate cancer with BRCA1 and BRCA2 founder mutations. Clin Cancer Res 2009;15(3):111220.

Ahmad, A., Wang, Z., Ali, R., Maitah, M. Y., Kong, D., Banerjee, S., et al. (2010). Apoptosis-inducing effect of garcinol is mediated by NF-kappaB signaling in breast cancer cells. J Cell Biochem, 109(6), 1134-1141.

Ahmed, F., Mahmud, S., Hatcher, J., & Khan, S. M. (2006). Breast cancer risk factor knowledge among nurses in teaching hospitals of Karachi, Pakistan: a cross-sectional study. BMC Nurs, 5, 6.

Alkhalaf, M. (2007a). Resveratrol-induced apoptosis is associated with activation of p53 and inhibition of protein translation in T47D human breast cancer cells. Pharmacology, 80(2-3), 134-143.