利用 SW620 細胞株探討傳統中草藥天然化合物抑制大腸直腸癌轉移能力之活性

Abstract

大腸直腸癌是目前全世界主要造成男性與女性因惡性腫瘤而死亡的癌症，根據行政院衛生署最新統計，大腸直腸癌之罹患率在台灣目前已攀升至第一位。然而，絕大多數癌症患者 (90%) 並非死於腫瘤本身，而是死於癌症轉移 (metastasis)。目前癌症治療方式以手術和化療為主，但仍有副作用和癒後復發等問題；隨著化學藥物療法受到限制，醫學轉而求助替代補充療法或傳統醫學治療，因此中草藥研發備受關注。 本研究即以一具有高度轉移特性之大腸直腸癌細胞株 SW620 作為體外篩選藥物之平台，並且回顧文獻選出 13 種具有抑制癌細胞轉移潛力之中草藥天然化合物，包含穿心蓮內酯 (Andrographolide)、吳茱萸鹼 (Evodiamine)、薑黃 (Curcumin)、泛黃芩素 (Wogonin)、黃芩素 (Baicalein)、黃芩苷 (Baicalin)、白楊素 (Chrysin)、雷公藤內酯 (Triptolide) 、雷公藤紅素 (Celastrol)、厚朴酚 (Magnolol)、異厚朴酚 (Honokiol)、薑辣素 (6-gingerol) 和薑烯酚 (6-shogaol)，藉此比較這些天然化合物抑制腫瘤轉移能力，並探討可能作用機制。 本實驗共可分作三階段。階段一，以 MTT assay 篩選出具有抑制癌細胞增生能力之天然化合物。在 13 種天然化合物中，有 7 種在 500 μM 以下可有效抑制 SW620 生長，其中以 curcumin (Cur) 和 honokiol (Hon) 效果最佳 (IC50 皆為 30 μM)，因此以 Cur 和 Hon 進入第二階段試驗。 階段二，分別以傷口癒合試驗 (Wound healing assay) 以及細胞侵入性試驗 (Boyden chamber assay) 評估天然化合物抑制腫瘤細胞爬行 (migration) 和入侵 (invasion) 能力。Cur 和 Hon 皆可有效抑制 SW620 爬行，並且呈現劑量效應；在入侵能力試驗部分，Cur 之抑制率由濃度低至高 (5, 10, 30 μM) 分別為 65%, 69% 和 93%，Hon 則為 40%, 70% 和 69%，以上結果顯示，Cur 和 Hon 皆具有效抑制癌細胞轉移之特性。 階段三，以西方點墨法 (Western blotting) 探討天然化合物抑制轉移之作用機制。實驗結果顯示，Cur 和 Hon 無法影響 MMP9 以及 MMP2 之蛋白質表現；但皆可以顯著降低活化態 (active form) 以及未活化 (pro-form) 之 MMP7 蛋白表現量，並且呈現劑量效應。另外，Cur 和 Hon 可以維持細胞膜上 E-cadherin，穩定細胞間黏著力，並抑制與 E-cadherin 形成 adheren junction 的 β-catenin 鬆脫，避免 β-catenin 轉入細胞核帶動下游癌化與轉移相關基因之表現，進而達到抑制細胞轉移之作用。

Colorectal cancer (CRC) is the major cancer which causes people dead worldwide. In Taiwan, according to the Bureau of Health Promotion, Department of Health, CRC has reached the first place in cancer incidence. Tumor cell metastasis is the most malignant feature during cancer progression and accounts for the major factor (90%) of mortality. Current cancer treatment includes surgery and chemotherapy, but there are still side effects and recurrence problems. Therefore, nowadays medicine profession pays more attention to the research and development of traditional Chinese herbal medicines (TCM). As a Consequence, our aim was to find the potential TCM to decrease the metastastic rates of colorectal cancer. In our study, we used the colorectal cancer cell line, SW620, which has high metastasis ability for in vitro assays. We also chose 13 pure compounds, which have the potential for anti-metastasis, from TCM based on the literatures to investigate the anti-metastastic ability of those pure compounds and the possible mechanism. The pure compounds include Andrographolide, Evodiamine, Curcumin, Wogonin, Baicalein, Baicalin, Chrysin, Triptolide, Celastrol, Magnolol, Honokiol, 6-gingerol and 6-shogaol. There are three phases of our experiments. In phase one, we tested the ability of anti-proliferation of those pure compounds by MTT assay. Then we selected curcumin (Cur) and honokiol (Hon), which are more effective than the others, to conduct bioactivity test. In the second phase, we used would-healing assay and Boyden chamber assay to analyze the potential of anti-migration and anti-invasion of Cur and Hon. Results showed that both of Cur and Hon had the inhibitory ability of anti-migration on SW620 in a dose-dependent manner. Besides, they also effectively reduced cell invasion. The inhibition rates of the treatments of different concentration of Cur (5, 10, 30μM) were 65%, 69% and 93%. For Hon, they were 40%, 70% and 69%. The data demonstrated that both of Cur and Hon could suppress the metastasis of SW620. Finally in phase three, we investigated the possible mechanism of the anti-migration effect of Cur and Hon. Using western blotting assay, we found that although Cur and Hon could not decrease MMP2 and MMP9 expression, they could significantly inhibit active and pro-form MMP7 protein expression with dose-dependency. Moreover, both of Cur and Hon could augment the expression and stability of E-cadherin proteins on the cell membrane to prevent the disruption of cell contacts. They could also avoid β-catenin to translocate into nuclear binding downstream targets such as MMPs (especially MMP7) thus downregulated cell metastasis. In conclusion, our findings suggest that Cur and Hon could maintain the expression of E-cadherin on cell membrane, avoid β-catenin translocation and then suppress the translation of MMP-7 protein, thus inhibit the metastasis of human colorectal cancer cell line SW620.