薑黃素及其結構類似物對於人類纖維肉瘤細胞 (HT-1080) 抑制轉移活性之相關研究 Studies on Curcumin and its analogs on anti-metastatic activities of human fibrosarcoma cells (HT-1080)
中文摘要
薑黃素 (curcumin)為存在於植物薑黃中的黃色色素，已被研究出具有抗發炎、抗癌症侵襲以及 抗轉移等多種活性。目前已有相當多研究試圖改變薑黃素的化學結構以合成生物活性優於薑黃 素的類似物。根據文獻，金屬基質蛋白酵素 (Matrix metalloproteinases, MMPs)以及尿激酶 型纖溶酶原激活劑 (urokinase-type plasminogen activator, uPA)在癌細胞進行轉移過程 對於細胞外間質的水解扮演關鍵的角色。本篇研究使用薑黃素及其二十三個結構類似物對於具 有高度轉移活性的人類纖維肉瘤細胞 (HT-1080)進行 MMP-9 活性、蛋白質及基因表現抑制活 性篩選，並探討其可能機制。除了二十四號化合物和三十一號化合物以2.5 μM 處理細胞之外，
薑黃素及其餘結構類似物以濃度5 μM 處理癌細胞並無顯著細胞毒性。由明膠酵素電泳法結果顯 示，二號化合物 (兩側苯環含有六個甲氧基取代)、七號化合物 (兩側苯環含有三個氫氧基、一個 甲氧基)與薑黃素在同濃度 5 μM 比較下具有較強抑制 MMP-9 活性的能力。本研究也同時以濃度 2.5 μM 的薑黃素、二號、七號、二十四號 (兩側苯環含有三個氫氧基取代)及三十一號化合物 (兩 側苯環含有兩個氫氧基，兩個甲氧基取代)處理 HT-1080 細胞，比較以上結構類似物對於 MMP-9 表現的影響。經由西方墨點法、反轉錄聚合酶連鎖反應結果顯示，二號、七號、二十四號 及三十一號化合物抑制MMP-9 基因的表現；由明膠酵素電泳法、纖維蛋白酵素電泳法及 MMP- 9 酵素活性試驗觀察到四個化合物能抑制 HT-1080 細胞的 MMP-9 活性，進而抑制了 HT-1080 細胞的遷移及侵襲能力。綜合以上，二號化合物和七號化合物比薑黃素、二十四號化合物和三十 一號化合物具有更好的抗轉移活性。第一型血紅素加氧酶 (Heme oxygenase 1, HO-1)，為 一種遭遇壓力便會誘導產生的蛋白質,且 HO-1 的誘導與 ROS 產生具有關連性，有研究指出其 可能為抑制MMPs 的原因。以西方墨點法觀察薑黃素、二號、七號、二十四號及三十一號化合物 對於HT-1080 細胞內的 HO-1 蛋白表現量，結果顯示薑黃素，二號化合物及三十一號化合物 可以顯著誘導HO-1 蛋白質的表現，然而七號化合物及二十四號化合物卻無法誘導之。以上結 果顯示，薑黃素、二號、七號、二十四號及三十一號化合物於體外試驗觀察到其對於癌細胞株遷 移、侵襲能力的抑制機轉可能來自於抑制MMP-9 基因表現和抑制 uPA 活性。另外，二號化合物 抑制MMP-9 活性的能力較佳推測有可能是其可顯著誘導細胞大量產生 HO-1 以對抗 PMA 所誘 導產生的ROS。由以上結果得知二號、七號、二十四號及三十一號化合物分別呈現不同程度抑制 MMP-9 及 uPA 且活性皆優於薑黃素。
英文摘要
Curcumin, a yellowish pigment existed in turmeric plant has been shown to exhibit numerous biological properties, such as anti-inflammation, antioxidation, anti-invasion, and antimetastatic activity. Chemical modifications of curcumin have been studied intensively in an attempt to find an analog with similar but enhanced biological activities of curcumin. In literatures, matrix
metalloproteinases (MMPs) and urokinase plasminogen activator (uPA) played important roles in cancer cell invasion by hydrolysing extracellular matrix (ECM).
In this study, curcumin and 23 novel analogs were used to screen for inhibition of matrix metalloproteinase-9 (MMP-9) activities, protein and gene expression of highly metastatic HT-1080 human fibrosarcoma cells to explore the mechanisms of action. All tested compounds, except analog 24 and analog 31 at concentration of 2.5 μM showed no cytotoxic activities at 5 μM. The analog 2 (six methoxy substitutes in phenyl groups) and analog 7 (three hydroxyl and one methoxyl substitutes in phenyl groups) showed higher MMP-9 inhibitory activities than curcumin did at 5 μM in gelatin zymography analysis. We comparatively examined the influence of analog 2, 7, 24 (three hydroxyl substitutes in phenyl groups), and 31(two hydroxyl and two methoxyl substitutes in phenyl groups) on the
expression of MMP-9 of HT-1080 cells at concentration of 2.5 μM. Analog 2, 7, 24 and 31 suppressed the gene expression of MMP-9 as the results of RT-PCR assay and Western blot assay, and decreased their corresponding activities in HT-1080 cells revealed by gelatin zymography assay, MMP-9 activity ELISA and fibrin zymography assay which resulted in inhibition of HT-1080 cell invasion and migration differentially. All in all, analog 2 and 7 showed higher anti-metastatic activities than analog 24 and analog 31 did. Heme oxygenase 1(HO-1), a stress- responsive enzyme, which was found to correlate with production of reactive oxygen species (ROS), suggesting a causative relationship on MMP inhibition.
Therefore, the effect of curcumin and analog 2, 7, 24 and 31 on HO-1 protein expression were also studied. Interestingly, exposure to curcumin, analog 2 and 31 treatment maximally induce HO-1 protein expression in HT-1080 cells, however, analog 7 and 24 were less apparently. These data suggested that the inhibitory effects of curcumin and analog 2, 7, 24 and 31 on MMP-9 gene
expression and uPA activity was closely related to tumor invasion and migration in vitro. Furthermore, analog 2 showed highly MMP-9 inhibitory activity which
possibly involved mechanisms related to its ability to induce HO-1 to suppress PMA-induced ROS, which can activate MMP-9 gene expression. In summary, these data demonstrated that analog 2, 7, 24 and 31 show higher anti-metastasis potency than curcumin by the differentially down-regulation of MMP-9 and uPA.
