Brazilein和Naphtho[1,2-b]furan-4,5-dione(NFD)對MDA-MB-231乳癌細胞抗轉移機制之研究

Antimetastatic mechanism of brazilein and naphtho[1,2-b]furan-4,5-dione (NFD) in MDA-MB-231 breast cancer cells

謝奇穎(Chi-Ying Hsieh)

指導教授 ： 林信仁

高雄醫學大學/生命科學院/醫藥暨應用化學研究所/碩士(2012年)

https://doi.org/10.6832/KMU.2012.00075

摘要

Brazilein，是一種從蘇木屬分離出的生物活性化合物，在東方民間常使用藥物。用劃痕實驗和Boyden chamber分析顯示無毒劑量的Brazilein處理細胞遷移和侵襲皆有顯著的抑制。Western blot, Reversed transcription-PCR 和Gelatin zymography檢測表明，Brazilein選擇性的抑制MMP-2的活性和蛋白表現。此外，Brazilein明顯降低NF-κB的蛋白表現，同時增加IκBα且阻止上游信號IKK的磷酸化。加入NF-κB抑制劑 (PDTC)，也抑制MMP-2的活性和細胞的遷移能力。依實驗結果顯示，Brazilein有效的抑制p38 MAPK蛋白，PI3K/Akt的磷酸化，同時不影響細胞外信號調節激酶 (ERK)1/2和c-Jun N-末端激酶 (JNK)的磷酸化。此外，用p38 MAPK抑制劑 (SB203580)或PI3K抑制劑 (Wortmannin)處理MDA-MB-231細胞，導致MMP-2的蛋白表現減少，且有明顯抑制細胞遷移。以上結果表明，Brazliein通過抑制PI3K/Akt和p38 MAPK信號通路和抑制NF-κB的活性，減少MMP-2的活性並抑制MDA-MB-231細胞的遷移和侵襲。
Naphtho[1,2-b]furan-4,5-dione (NFD)，是由海茄苳屬Avicennaceae家族Avicennia marina分離出之生物活性成分，且已被證明具有抗癌活性。在各種腫瘤中，表皮生長因子受體 (EGFR)的激活誘導信號通路與腫瘤轉移有關，包括乳癌。我們使用表皮生長因子 (EGF)誘導MDA-MB-231細胞並探討NFD在細胞轉移上的影響。NFD在無明顯細胞毒性劑量下抑制細胞遷移和侵襲，降低c-Jun和c-Fos的蛋白表現，且降低MMP-9表現和活性。NFD抑制EGF誘導EGFR和磷酸肌醇3-激酶 (PI3K)/Akt的磷酸化。特定的PI3K抑制劑Wortmannin，明顯阻斷EGF誘導細胞遷移和侵襲。此外，EGFR抑制劑AG1478抑制EGF誘導MMP-9的表現和細胞的轉移，以及PI3K/Akt的活性，顯示NFD抑制活化EGFR，導致PI3K/Akt的去活化。這些結果顯示，NFD抑制EGF誘導MDA-MB-231細胞侵襲和遷移通過EGFR所屬PI3K/Akt信號，導致MMP-9表現下降。這些結果提供NFD對EGF刺激MDA-MB-231細胞抗轉移分子作用機制。

關鍵字

表皮生長因子/表皮生長因子受體 ； 遷移

並列摘要

Brazilein, a bioactive compound isolated from Caesalpinia sappan L., has long been used in oriental folk medicines. During treatment with non-toxic doses of brazilein, cell migration and invasion were markedly suppressed using wound-healing assay and Boyden chamber assay. Western blot, reversed transcription-PCR and gelatin zymography analysis showed that brazilein significantly and selectively suppressed the expression and activity of MMP-2 in a concentration-dependent manner. In addition, brazilein significantly decreased the nuclear level of NF-κB, increased inhibitor of kappa Bα (IκBα) through preventing phosphorylation of upstream signal IκB kinase (IKK), and pretreatment with a specific NF-κB inhibitor PDTC also reduced MMP-2 activity and cell migration. Our biochemical assays indicated that brazilein potently suppressed the phosphorylation of p38 MAPK, phosphatidylinositide-3-kinase (PI3K) and Akt, while it did not affect phosphorylation of extracellular signal regulating kinase (ERK)1/2 and c-Jun N-terminal kinase (JNK). Additionally, the treatment of inhibitors specific for p38 MAPK (SB203580) or PI3K (wortmannin) to MDA-MB-231 cells could result in a reduced expression of MMP-2, concomitantly with a marked inhibition on cell migration. Taken together, these results demonstrate that brazilein inhibits the migration and invasion of MDA-MB-231 cells by reducing MMP-2 activity through suppressing PI3K/Akt and p38 MAPK signaling pathways and inhibiting NF-κB activity.
Naphtho[1,2-b]furan-4,5-dione (NFD), a bioactive component of Avicennia marina, has been demonstrated to display anti-cancer activity. Activation of epidermal growth factor receptor (EGFR)-induced signaling pathway has been correlated with cancer metastasis in various tumors, including breast carcinoma. We use EGF as a metastatic inducer of MDA-MB-231 cells to investigate the effect of NFD on cell metastasis. NFD suppressed EGF-mediated protein levels of c-Jun and c-Fos, and reduced MMP-9 expression and activity, concomitantly with a marked inhibition on cell migration and invasion without obvious cellular cytotoxicity. NFD abrogated EGF-induced phosphorylation of EGF receptor (EGFR) and phosphatidylinositol 3-kinase (PI3K)/Akt. The specific PI3K inhibitor, wortmannin, significantly blocked EGF-induced cell migration and invasion. Furthermore, the EGFR inhibitor AG1478 inhibited EGF-induced MMP-9 expression and cell metastasis, as well as the activation of PI3K/Akt, suggesting that PI3K/Akt activation occur downstream of EGFR activation. These findings suggest that NFD inhibited the EGF-induced invasion and migration of MDA-MB-231 cells via EGFR-dependent PI3K/Akt signaling, leading to the down-regulation of MMP-9 expression. These results provide a novel mechanism to explain the role of NFD as a potent anti-metastatic agent in MDA-MB-231 cells.

並列關鍵字

Brazilein ； NFD ； EGF/EGFR ； migration ； MDA-MB-231 cells ； MMP-2 ； p38 MAPK ； PI3K/Akt

參考文獻

Alessi, D.R., Andjelkovic, M., Caudwell, B., Cron, P., Morrice, N., Cohen, P., Hemmings, B.A., 1996. Mechanism of activation of protein kinase B by insulin and IGF-1. EMBO Journal 15, 6541-6551.

Alper, O., Bergmann-Leitner, E.S., Bennett, T.A., Hacker, N.F., Stromberg, K., Stetler-Stevenson, W.G., 2001. Epidermal growth factor receptor signaling and the invasive phenotype of ovarian carcinoma cells. Journal of the National Cancer Institute 93, 1375-1384.

Bahassi, E.M., Karyala, S., Tomlinson, C.R., Sartor, M.A., Medvedovic, M., Hennigan, R.F., 2004. Critical regulation of genes for tumor cell migration by AP-1. Clinical & Experimental Metastasis 21, 293-304.

Baselga, J., Arteaga, C.L., 2005. Critical update and emerging trends in epidermal growth factor receptor targeting in cancer. Journal of Clinical Oncology 23, 2445-2459.

Bernhard, E.J., Gruber, S.B., Muschel, R.J., 1994. Direct evidence linking expression of matrix metalloproteinase 9 (92-kDa gelatinase/collagenase) to the metastatic phenotype in transformed rat embryo cells. Proceedings of the National Academy of Sciences of the United States of America 91, 4293-4297.