天然物抑制癌瘤細胞轉移及誘導細胞凋亡機制探討

Study of the effects and mechanisms of natural compounds on cancer cell metastasis and cell apoptosis

陳霈霓(Pei-Ni Chen)

指導教授 ： 謝易修

中山醫學大學/醫學院/生化暨生物科技研究所/博士(2007年)

https://doi.org/10.6834/CSMU.2007.00117

摘要

癌細胞的轉移往往是造成癌症病人病灶復發致死及最難根治的主要原因，其過程包括細胞的貼附、細胞外基質的降解及細胞轉移能力的改變。花青素與類黃酮是廣存於各類蔬果中之主要成分，目前已有許多文獻指出其具有抗氧化活性、降低血脂肪、促使癌瘤細胞凋亡及防止正常細胞癌化等作用。此外許多莓果及花朵中也含有豐富的花青素與類黃酮，其萃取物已被應用於治療許多疾病。在本篇論文中，我們首次證實由黑糯米所萃取的花青素(Oryza sativa L. anthocyanins (OAs)、cyanidin 3-glucoside與peonidin 3-glucoside)和桑椹所萃取的花青素(mulberry anthocyanins (MACs)、cyanidin 3-rutinoside與cyanidin 3-glucoside) 可顯著抑制具有高度轉移能力之人類肺癌細胞(A549與H1299)與老鼠肺癌細胞(Lewis lung carcinoma; LLC)的侵犯及移動能力，利用gelatin-及casein-zymography分析方法，探討細胞外基質降解相關基因，如matrix metalloproteinase-2 (MMP-2)、matrix metalloproteinase-9 (MMP-9)及urokinase-type plasminogen activator (u-PA)的活性表現，結果發現OAs、MACs、cyanidin 3-glucoside、peonidin 3-glucoside與cyanidin 3-rutinoside對MMP-2、MMP-9與u-PA的分泌量有明顯的抑制，並呈現濃度的效應關係，此外cyanidin 3-glucoside也可明顯促進A549細胞tissue inhibitor matrix metalloproteinase-2 (TIMP-2)與plasminogen activator inhibitor-1 (PAI-1)之蛋白表現，進一步利用reverse transcription-polymerase chain reaction (RT-PCR)，其MMP-2、MMP-9、u-PA、TIMP-2與PAI-1的mRNA表現變化與蛋白表現量一致，顯示這些水解酶相關基因是調控在mRNA的層次，深入探討其作用機轉，結果顯示cyanidin 3-glucoside、peonidin 3-glucoside可顯著抑制p-ERK1/2、c-Jun與NF-κB 的表達，
進而抑制AP-1 和NF-κB 與DNA 的結合能力，實驗中利用H1299 細
胞轉殖p-ERK1/2 的上游MEK1，使其大量表達，促使p-ERK1/2 可
以持續磷酸化，當細胞轉殖持續磷酸化之MEK1 會使得細胞的移動
能力明顯上升，但是當處理peonidin 3-glucoside與cyanidin 3-glucoside 並無法抑制由持續磷酸化之MEK1 所誘導之細胞侵襲能力，顯示peonidin 3-glucoside 與cyanidin 3-glucoside 可能是作用於p-ERK1/2 的上游。以silibinin 處理人類口腔癌細胞(SCC-4)時，可顯著抑制其細胞的侵犯及移動能力，其作用可能是經由抑制細胞MMP-2 與u-PA 之分泌，並且促進其內生性抑制劑TIMP-2 與PAI-1 之蛋白與mRNA 表現，進一步利用RT-PCR，MMP-2、u-PA、TIMP-2 與PAI-1 的mRNA 表現量與蛋白表現呈一致的變化，探討其訊息傳遞路徑，發現當SCC-4 處理silibinin 其可顯著抑制p-ERK、c-Fos、c-Jun 與NF-κB 的表達。利用乳癌細胞(Hs578T)處理低濃度cyanidin 3-glucoside 與peonidin 3-glucoside 時則會經由讓細胞週期停滯在G2/M 時期進而抑制細胞的增生，並且在高濃度時則會誘導使其走向細胞凋亡。最後在動物實驗中， 我們利用C57BL/6 老鼠在皮下種植Lewis lung carcinoma，發現當老鼠給予silibinin、OAs、cyanidin 3-glucoside 與peonidin 3-glucoside 會抑制腫瘤大小與癌細胞轉移到肺部的能力，此外將人類肺癌細胞異種移植於裸鼠皮下，當處理cyanidin 3-glucoside 與peonidin 3-glucoside，則會明顯抑制腫瘤的大小。綜合以上結果，
推測silibinin、cyanidin 3-rutinoside、cyanidin 3-glucoside 或peonidin 3-glucoside 可能是透過抑制腫瘤細胞株MMPs 及u-PA 分泌作用進而抑制癌瘤細胞遷移和侵犯作用的能力，而cyanidin 3-glucoside 與 peonidin 3-glucoside 則是可以抑制乳癌細胞增生，且進一步誘導細胞走向凋謝死亡，我們期望以上結果日後或許可應用於癌症的臨床輔助治療。

關鍵字

轉移 ； 花青素 ； 侵襲 ； 基質金屬蛋白酶

並列摘要

Metastasis, the major cause of cancer death and various treatment strategies have targeted on preventing the occurrence of metastasis, is a multi-step process involving cell adhesion and proteolytic degradation of the extracellular matrix (ECM), essential to achieving cell motility. Many bioactive properties of anthocyanins and flavonoid, present in various fruits and vegetables as natural colorants, have been well characterized. They are widely used for their antioxidant properties. Recent studies have also revealed pleiotropic anticancer and antiproliferative capabilities of anthocyanins. Berry extract contains high amounts of anthocyanins and is commonly used in certain diet and therapeutic applications. This study first demonstrates that, in the absence of cytotoxicity, black rice anthocyanoins (Oryza sativa L. anthocyanins (OAs), peonidin 3-glucoside, and cyanidin 3-glucoside), mulberry anthocyanins (mulberry anthocyanins (MACs), cyanidin 3-rutinoside, and cyanidin 3-glucoside), or silibinin exerted a dose-dependent inhibitory effect on the invasion, and motility of highly metastatic human lung cancer cells (A549 and H1299) and murine lung cancer cells (Lewis lung carcinoma; LLC). We examined the effect of anthocyanins on factors of cancer metastasis. We treated tumor cells with various concentrations of anthocyanins, for set periods, and then subjected cells to gelatin zymography, casein zymography, and Western blot to investigate the expression of matrix metalloproteinase-2 (MMP-2), matrix metalloproteinase-9 (MMP-9), urokinase-type plasminogen activator (u-PA), tissue inhibitor matrix metalloproteinase -2 (TIMP-2), and plasminogen activator inhibitor-1 (PAI-1). Following treatment with these compounds was found to decrease the expression of MMP-2, MMP-9, and u-PA in a concentration-dependent manner and enhance the expression of TIMP-2 and PAI-1. Further analysis with semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) showed that these alternations were all on the transcriptional levels. Further, a treatment of cyanidin 3-glucoside, peonidin 3-glucoside, or cyanidin 3-rutinoside also resulted in an inhibition on the activation of p-ERK1/2, c-Jun, c-Fos, or NF-κB and decreases the DNA binding activity. Overexpression with MEK1 blocked peonidin 3-glucoside and cyanidin 3 glucoside-inhibited H1299 cell invasion, amd suggests that the target point is the upstream of ERK 1/2. We also demonstrated that silibinin could significantly inhibit the invasion, motility, secretion of MMP-2 or u-PA of oral squamous cell
carcinoma (SCC-4) together with an enhanced expression of TIMP-2 and PAI-1. A treatment with silibinin also led to a dose-dependent inhibition on the activation of ERK1/2, NF-κB, c-Jun and c-Fos. Cyanidin 3-glucoside and peonidin 3-glucoside may exert a cell growth inhibitory through an arrest of G2/M phase of cell cycle, inhibition of cell
proliferation and induction of apoptosis in Hs578T human breast carcinoma cells. Treatment for OAs, cyanidin 3-glucoside, peonidin 3-glucoside, or silibinin suppressed the metastasis and tumor volume of LLC cells in C57BL/6 mice. Moreover, peonidin 3-glucoside and cyanidin 3-glucoside suppressed the tumor growth in H1299 xenograft
nude moce. Together, these results suggest that cyanidin 3-glucoside, peonidin 3-glucoside, or silibinin may act to decrease the in vitro protease expression of cancer cells and therefore, reduce the invasion and metastasis of tumor cells, while cyanidin 3-glucoside and peonidin 3-glucoside could inhibit breast cancer cells proliferation, and induce cell apoptosis. This study suggests a possible role for anthocyanins in cancer therapy.

並列關鍵字

metastasis ； anthocyanins ； invasion ； MMP

參考文獻

Wang, Q., Han, P., Zhang, M., Xia, M., Zhu, H., Ma, J., Hou, M., Tang, Z., and Ling, W. (2007): Supplementation of black rice pigment fraction improves antioxidant and anti-inflammatory status in patients with coronary heart disease. Asia Pacific Journal of Clinical Nutrition 16 Suppl 1, 295-301.

Collen, D. (1980): On the regulation and control of fibrinolysis. Edward Kowalski Memorial Lecture. Thrombosis and Haemostasis 43, 77-89.

Liabakk, N. B., Talbot, I., Smith, R. A., Wilkinson, K., and Balkwill, F. (1996): Matrix metalloprotease 2 (MMP-2) and matrix metalloprotease 9 (MMP-9) type IV collagenases in colorectal cancer. Cancer Research 56, 190-6.

Singh, R. P., Dhanalakshmi, S., Tyagi, A. K., Chan, D. C., Agarwal, C., and Agarwal, R. (2002): Dietary feeding of silibinin inhibits advance human prostate carcinoma growth in athymic nude mice and increases plasma insulin-like growth factor-binding protein-3 levels. Cancer Research 62, 3063-9.

Taylor, W. R., DePrimo, S. E., Agarwal, A., Agarwal, M. L., Schonthal, A. H., Katula, K. S., and Stark, G. R. (1999): Mechanisms of G2 arrest in response to overexpression of p53. Molecular Biology of the Cell 10, 3607-22.