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題名 | 抗肝癌方劑之資源開發與作用機轉之探 討:傳統方劑對人類肝癌細胞之體內及體 外抑制增生、腫瘤入侵、血管新生作用和誘發細胞程式死亡機制之探討(全程總報告)=The Mechanism of the Anti-proliferative, Anti-invasive, Anti-angiogenesis and Apoptotic Effects of Traditional Chinese Medicinal Prescription (TCMP) in Human Liver Cancer Cells in Vitro and in Vivo. (Final Report) |
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作者 | 林俊清; | 書刊名 | 中醫藥年報 |
卷期 | 28:7 2010.09[民99.09] |
頁次 | 頁195-266 |
分類號 | 414.32 |
關鍵詞 | 黃連解毒湯; 肝癌; 細胞凋亡; 腫瘤入侵; 移行; NF-κB; Glabridin; ERK1/2; Huang-Lian-Jie-Du-Tang; Liver cancer; Apoptosis; NF-κB; |
語文 | 中文(Chinese) |
中文摘要 | 本研究目標乃從生命科學發展迅速的分子生物學理論與傳統中國醫藥方 劑之作用相互配合,深入探索其抗肝癌之活性機轉,如此不僅可增加西方醫 學對中國醫藥作用的接受度,並賦與中國醫藥複方藥效的現代化意義。癌症 目前為威脅人類健康的頭號敵人,雖然全世界各國都投入大量的資源進行研 究,但目前仍有許多困難仍需克服:其中包括許多化療藥物上不能完全殲滅 癌症細胞。再者,對於一些具轉移性及複發性的癌細胞會發展出高度的抗藥 性,造成治療的失敗。除此,透過血管新生的作用,癌細胞會隨血行轉移至 他處,而造成癌細胞的快速擴散進而造成病人的死亡。 藥物對細胞增生的抑制活性以XTT方式測得;細胞週期的分佈則是以核 酸染料將DNA染色後以flow cytometry的方式分析;Apoptosis的偵測以瓊膠電泳和TUNEL方法分析;粒腺體膜電位以JC-1染色。p21的表現以ELISA方式測得;caspase的活性以caspase activity assay kit分析。NF-κB活性以TransAM ELISA kit分析。其他蛋白質的表現以Western blot的方式偵測;in vivo的活性 評估以nude mice的實驗方式評估。 細胞移行活性以QCM™ 24-well Cell Migration Assay和傷口癒合試驗分 析。細胞入侵能力以BD BioCoat Tumor Invasion System分析。MMP和VEGF分泌量以ELISA分析。MMP活性以Gelatin zymography分析。各種蛋白質表現以Westen blot分析。AP-1活性以EMSA和report plasmid分析。基因以siRNA做阻斷。動物血管新生作用以Matrigel Plug Angiogenesis Assay研究發現傳統方劑 黃連解毒湯和散腫潰堅湯對人類肝癌細胞株HepG2和PLC/PRF/5細胞具有良好 的細胞增生抑制效果。在濃度500 μg/ml的濃度可達最好的增生抑制效果。黃連解毒湯和散腫潰堅湯對抗HepG2細胞增生的IC50值分別為192.3和303.4μg/ml;對抗PLC/PRF/5細胞增生的IC50值分別為160.9和333.2 μg/ml。 在黃連解毒湯的作用機轉方面,細胞以黃連解毒處理12小時後可促使HepG2和PLC/PRF/5的細胞週期停滯於S-G2/M期,此一效果隨著劑量增加而 增加。再者,以瓊膠電泳和TUNEL染色的方法也發現黃連解毒湯會誘發細 胞進行細胞凋亡,此一效果隨著劑量和處理時間增加而增加。在分子機制方 面,黃連解毒湯湯可以透過p53-independent的方式增加p21的表現。黃連解毒湯也會抑制cyclin A、cyclin B、cdc2、cdc25C的表現以及促使cdc2和cdc25保持於不活化的磷酸化狀態,進而抑制細胞週期的演進。再者,黃連解毒 湯也會改變proapoptotic和antiapoptotic Bcl-2蛋白的比值進而啟動粒線體相關 的apoptosis路徑,包括粒腺體膜電位的改變、caspase-9的活化而造成細胞死 亡。相較於細胞細胞死亡路徑,黃連解毒湯也會抑制細胞生存路徑NF-κB 的轉位、活性以及下游分子Bcl-XL的表現而促進細胞的死亡。最後,利用in vivo的實驗模式也證實黃連解毒湯在動物體也具有腫瘤抑制的效果。 第二年計畫以評估傳統方剤及活性成分對腫瘤移行及入侵能力的抑制 作用為主軸。初步結果發現散腫潰堅湯和黃連解毒皆可抑制肝癌細胞株Hep G2和PLC/PRF/5的腫瘤入侵(invasion)作用情形,且散腫潰堅湯比黃連解毒 的作用更明顯。接續以散腫潰堅湯與黃連解毒湯的單味組成其所含的純化物(pure compound)在濃度50μM時對肝癌細胞株Hep G2和PLC/PRF/5的腫瘤入侵(invasion)作用。篩選成分黃芩(Baicalin)、黃芩素(Baicalein)、漢黃芩素 (Wogonin)、小蘗鹼(Berberine)、黃連鹼(Coptisine)、去羥梔子甘(Geniposide)、 龍膽苦苷(Gentiopicroside)、柴胡皂甙 d (Saikosaponin d)、甘草酸(Glycyrrhizic acid)、光甘草定(Glabridin)、薑黃素(Curcurmin)、齊墩果酸(Oleanolic acid)、 白樺脂酸(Betulinic acid)、葛根素(Puerarin)、芍藥甘(Paeoniflorin),芍藥內 酯苷(Albiflorin)。其中包括黃芩素(Baicalein)、薑黃素(Curcurmin)、齊墩果酸(Oleanolic acid)、光甘草定(Glabridin)等具有明顯的抑制肝癌腫瘤入侵(invasion)作用。其中又以光甘草定(Glabridin)效果最佳。因此以光甘草定(Glabridin)做深入之探討。 光甘草定可以有效抑制肝癌細胞株HepG2和LC/PRF/5肝癌細胞的移行和入侵,且具有劑量依賴的效果。再者,研究亦證實光甘草定不但可有效抑 制和癌細胞轉移的相關MMP-9的表現及活性,亦可降低和血管新生相關分子 VEGF的表現。在分子機轉方面,光甘草定會抑制ERK1/2的活化,並降低其 下游分子Elk-1由細胞質轉位至細胞核中及抑制Elk-1在細胞核內的磷酸化。透過抑制ERK1/2的活化,光甘草定也會抑制AP-1轉錄因子的DNA結合能力及 促進基因轉錄活性。再者,當細胞以siRNA技術將ERK2做部分阻斷時,發現 ERK siRNA的轉殖可加強光甘草定對肝癌細胞的移行和入侵的抑制效果,因 此推論光甘草定是透過抑制ERK1/2的活化進而將低肝癌細胞的移行和入侵。最後,本研究亦以Matrigel plug angiogenesis assay分析光甘草定在動物體內是 否具有抑制血管新生。結果發現,光甘草定可效抑制PLC/PRF/5引起德血管 新生以及腫瘤的生長。因此本計畫證實甘草活性組成光甘草定具可潛在價值 開發為抗肝癌轉移的有效化合物。 |
英文摘要 | The incidence of cancer is increasing worldwide and it is the single most common cause of deaths in both developed and developing countries. Hepatocellular carcinoma (HCC) is one of the most lethal malignancies, and is also one of the four most prevalent malignant diseases of adults in China, Taiwan, Korea, and sub-Africa. Several etiologic factors have been classified as high-risk factor in association with HCC, including exposure to aflatoxin B1, and infection with hepatitis B virus and hepatitis C virus.Our laboratory focuses on the integration of traditional Chinese traditional medicines (CTM) with molecular biology to further study the active mechanism their anti-liver cancer activity. This allows a better appreciation of CTM in the modern era and also a better understanding of its underlying potential for therapy, thus increasing its acceptance in Western medicine. Cell proliferation inhibition was assay by XTT. Cell cycle distribution was determined by flow cytomatry. Quantitative assessment of apoptosis was analyzed by agrose electroporsis and TUNEL method. The p21 level was assay by ELISA kits. The activity of caspases was measured by caspase activity assay kits. The mitochondraoal membrane potential was assessed by JC-1. The activity of NF-κB was assessed by Trans AM ELISA kit. The tumor growth of SZKJT was assessed by In vivo tumor xenograft study. Cell migration was assessed by QCM™ 24-well Cell Migration Assay and Scratch wound–healing assay. Cell invasion was assessed by BD BioCoat Tumor Invasion System. The expression of MMP and VEGF was assessed by ELISA. The MMP activity was determined by zymography. The activity of AP-1 was measured by EMSA and report plasmid transfection. The knockdown of ERK was carried out by siRNA tansfection. Our study reports here for the first time the anticancer effect of HLJDT in two human liver cancer cell lines, Hep G2 and PLC/PRF/5. The results indicated that HLJDT inhibited the proliferation of Hep G2 and PLC/PRF/5 cells by inducing apoptosis and blocking cell cycle progression in the S-G2/M phase. Immunoblot assay showed that HLJDT significantly increased the expression of inactivated phospho-Cdc2 protein and phospho-Cdc25C, and decreased the levels of Cyclin A, Cyclin B1, Cdc2, and Cdc25C, thereby contributing to the cell cycle arrest. Apoptosis induced by HLJDT in both Hep G2 and PLC/PRF/5 cells was determined by electrophoresis and TUNEL assay. In investigating the detailed mechanism, HLJDT increased the expression of Bax and Bak, but decreased the level of Bcl-2 and Bcl-XL, and subsequently triggered the mitochondrial apoptotic pathway (mitochondrial membrane potential loss and caspase-9 activation). In addition, HLJDT also inhibited cell survival signaling by enhancing the amount of IκBα in the cytoplasm, reducing the level and activity of NF-κB in the nucleus, and subsequently attenuating the expression of Bcl-XL in Hep G2 and PLC/PRF/5 cells. The inhibitory effect mediated by HLJDT on cell growth was also demonstrated in a nude mouse model, in which the liver cancer cells-induced tumor xenograft shrunk considerably following treatment with HLJDT. Taken together, these results suggest a potential anticancer effect of HLJDT against human liver cancer cells. In this plan, we first found that San Zhong Kui Jian Tang (散腫潰堅湯) had inhibitory effect on breast cancer cells invasion. Therefore, we assessed various active constituent of San Zhong Kui Jian Tang ingredients, including baicalin, baicalein, wogonin, berberine, coptisine, geniposide, gentiopicroside, saikosaponin d, curcurmin, oleanolic acid, puerarin, paeoniflorin, albiflorin and glabridin. From preliminary research and screening, glabridin exhibited a significant effect on liver cancer invasion. Next, we focus on the anti-migration and anti-invasion effect of glabridin, a flavonoid obtained from licorice, in the two human liver cancer cell lines Hep G2 and PLC/PRF/5. Treatment with glabridin decreased the cancer migration and invasion of the liver cancer cells in a dose dependent manner. This effect was strongly associated with a concomitant decrease in either the level or activity of MMP-9and VEGF. Glabridin inhibited both the activation and activity of ERK1/2 following a decrease of Elk-1 phosphorylation in the nuclei. In addition, glabridin also decreased the DNA binding and transcriptional activity of AP-1. Inhibition of ERK2 expression by specific ERK2 siRNA reinforces glabridin-mediated inhibition of cancer migration and invasion. More importantly, glabridin also exhibited an inhibition effect on angiogenesis in a Matrigel plug angiogenesis assay. Our results indicate that glabridin inhibits the activation of ERK1/2/AP-1, and may provide a molecular basis for drug development in the prevention and treatment of cancer. |
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