順、逆式肉桂酸生理功效之探討 (I) 對人類肺癌細胞侵入轉移性之抑制功效 (II) 對多重抗藥性結核菌生長抑制與藥物協同功效

第一部分　順、逆式肉桂酸生理功效之探討　(I)　對人類肺癌細胞侵入轉移性之抑制功效肉桂酸 (3-phenyl-2-propenoic acid, cinnamic acid, CA) 存在於各類植物性食品中，以逆式 (trans-) 肉桂酸為主要型式；文獻指出逆式肉桂酸具有抗發炎、抗氧化、及抑制癌細胞生長的生理功效。順式 (cis-) 肉桂酸在自然界含量稀少而取得不易，有研究發現順式肉桂酸比逆式肉桂酸具有較佳的生理活性，但對癌細胞侵入轉移的抑制功效方面卻仍未深入探討。肺癌是一種致命性的癌症，其中癌細胞轉移是造成約 70% 肺癌病患死亡的主因。因此，抑制肺癌細胞發生轉移是延長病人生命重要的一環。基質金屬蛋白酶 (matrix metalloproteinase, MMP) -2 及 -9 是與癌症侵入轉移最有關聯性的分解酶，若能有效抑制細胞中 MMP-2 和 -9 的活性，應能間接抑制細胞轉移的發生。本研究的目的為分析比較順式與逆式肉桂酸抑制人類肺癌細胞侵入轉移性之功效與作用機制。以不同濃度 (0, 25, 50, 100, 150, and 200 M) 順式及逆式肉桂酸處理經 phorbol-12-myristate-13-acetate (PMA) 誘發之 A549 細胞，於 37 ℃培養 24 小時後，以 casein zymography 測定細胞中 urokinase-type plasminogen activator (uPA) 的活性，以 gelatin zymography 測定細胞中 MMP-2 與 -9 的活性，進一步以 Reverse Transcription Polymerase Chain Reaction (RT-PCR) 評估 MMP-2 和 -9 的 mRNA 表現，並分析細胞黏附力、移行性及侵入性的大小，最後利用 Western blotting 評估順式及逆式肉桂酸抑制 A549 細胞 MMP-2 及 -9 的相關分子機轉。結果發現，順、逆式肉桂酸均以劑量效應方式抑制 PMA 誘發的 uPA、MMP-2 和 -9 的活性；順、逆式肉桂酸對 A549 細胞黏附力無顯著抑制作用；逆式肉桂酸可降低 MMP-2 之 mRNA 表現量，順式肉桂酸同時降低 MMP-2 和 -9 的 mRNA 表現量。在細胞傷口癒合與移行的試驗中，順式與逆式肉桂酸在濃度高於 50 M 時皆有抑制 A549 細胞移行的功效，相同劑量下，同樣也有抑制 A549 細胞侵入的作用，此抑制作用可能源自於訊息傳遞路徑 MAPKs 磷酸化的調控，而細胞核內之轉錄因子 AP-1 (c-Jun) 及 NF-B (p65) 蛋白質含量亦會隨作用濃度增加而逐漸減少。由以上結果得知，順、逆式肉桂酸皆能抑制 A549 細胞侵入作用，而順式肉桂酸比逆式肉桂酸有更高的抗侵入活性。順式肉桂酸的抗侵入功效可能藉由抑制 MAPKs 訊息傳遞路徑並抑制轉錄因子 AP-1 (c-Jun) 與 NF-B (p65) 的活化，降低 MMP-2 和 -9 之 mRNA 表現量，同時降低 uPA、MMP-2 及 -9 酵素活性，降低細胞的移行能力所致。第二部分　順、逆式肉桂酸生理功效之探討　(II) 對多重抗藥性結核菌生長抑制與藥物協同功效　　結核病 (Tuberculosis, TB) 是全球最嚴重的傳染病之一，長期使用不同抗生素組合治療的過程中容易導致一些不利的副作用，若病人不當服用藥物則會導致多重抗藥性結核病 (multiple-drug resistant tuberculosis, MDR-TB) 的發生。肉桂酸 (cinnamic acid) 是一種具抗菌作用的天然多酚類化合物，在植物界裡有順式 (cis-) 與逆式 (trans-) 兩種結構異構物，順式肉桂酸 (cis-CA) 可以在陽光照射下轉換成逆式肉桂酸 (trans-CA)。由於順式肉桂酸取得不易，關於順式肉桂酸生理功效的研究文獻仍相當有限。根據文獻指出逆式肉桂酸具有抗真菌、抗氧化、抑制癌細胞生長的生理功效；而順式肉桂酸抗真菌活性較逆式肉桂酸高，抗氧化活性也較逆式肉桂酸佳。Mycobacterium tuberculosis 是造成感染結核病的主要菌株，其中合成細胞壁的主要酵素為 uridine diphosphate-glucose pyrophosphorylase (UGPase；EC 2.7.7.9)，因此，如果能發展有效的增效劑或抑制 UGPase 表現量使細胞壁無法合成，將可使菌無法存活。本研究針對順式及逆式肉桂酸與第一線抗結核菌藥物isoniazid (INH) 與rifampicin (RIF) 對抑制多重抗藥性 M. tuberculosis生長的協同作用 (synergistic effects)，以及對 UGPase 表現量之抑制功效進行探討。結果顯示，順式與逆式肉桂酸均有抑制多重抗藥性結核菌生長的能力，但是順式肉桂酸之抑菌功效約為逆式肉桂酸的 120 倍。同時，順式及逆式肉桂酸與 INH 及 RIF 共用時，具有抑制菌體生長的協同作用。此外，經由掃描式電子顯微鏡觀察發現，順式肉桂酸確實具有破壞多重抗藥性結核菌細胞壁的能力。Polymerase Chain Reaction (PCR) 結果顯示，多重抗藥性 M. tuberculosis 中確實具有 ugp 基因存在；而以 Reverse Transcription Polymerase Chain Reaction (RT-PCR) 分析菌體中 UGPase mRNA 表現量，同樣發現順式肉桂酸比逆式肉桂酸具有明顯抑制效果。總結來說，順式肉桂酸比逆式肉桂酸具有明顯抑制多重抗藥性 M. tuberculosis生長與藥物協同功效，其機制可能藉由抑制 UGPase 表現量使結核菌細胞壁無法生合成，而致使菌體無法生長。

關鍵字

肉桂酸；基質金屬蛋白酶；人類肺癌細胞；侵入；轉移；結核病；多重抗藥性結核菌

並列摘要

Biological functions of cis- and trans-cinnamic acid (I) Inhibition on invasion and metastasis of human lung carcinoma cells 　　Cinnamic acid (3-phenyl-2-propenoic acid, CA) is a natural compound with several isomers that presented in various plant derived foods, and trans-cinnamic acid (trans-CA) is the predominant form. Although numerous pharmacological effects of trans-CA, such as anti-inflammation, anti-oxidation, and anti-cancer, have been found, cis-cinnamic acid (cis-CA) is considered possesses a higher physiological activity than trans-CA.　Lung cancer is a lethal cancer type and the major cause of tumor-related death. Approximately 70% of lung cancer patients die from metastasis. Therefore, treatments that limit the spread of tumors to new sites and blockade their invasion have been pursued to enhance the survival of cancer patients. Matrix metalloproteinase (MMP) -2 and -9, two proteolytic enzymes that highly expressed in various malignant tumors, are presumed to be associated with the progression and invasion of several cancer cells. It is commonly considered that the inhibition of MMP-2 or -9 activities to be an effective strategy for the prevention of tumor invasion and metastasis. 　　The purpose of this study was to evaluate the inhibitory effect of cis-CA and trans-CA on the invasion and metastasis of A549 cells. Treating the cells with various concentrations (0, 25, 50, 100, 150, or 200 M) of cis-CA and trans-CA in the presence 200 nM phorbol-12-myristate-13-acetate (PMA) at 37C for 24 h. The urokinase-type plasminogen activator (uPA) activities were determined by casein zymography, the MMP-2 and -9 activities were determined by gelatin zymography, and the mRNA levels of MMP-2 and -9 were investigated by Reverse Transcription Polymerase Chain Reaction (RT-PCR) assays; cell-matrix adhesion, migration, and invasion activities were also analyzed. The results showed that the treatment of cis- and trans-CA reduced the PMA-induced uPA, MMP-2 and -9 activities in a dose-dependent manner but has no significant effect on the adhesive activity of cells. The mRNA level of MMP-2 was inhibited by trans-CA, but both the mRNA levels of MMP-2 and -9 were inhibited by cis-CA. According to the results of wound healing and motility assays, the migration activity of PMA-induced A549 cells was decreased after treating with cis- and trans-CA at a concentration higher than 50 M. The PMA-induced invasion activity of A549 cells were also suppressed by cis- and trans-CA at the same dose used in motility assay. The signaling of MAPKs and the transcription factor of AP-1 (c-Jun) and NF-B (p65) in A549 cells were also inhibited by cis- and trans-CA. In conclusion, cis- and trans-CA are inhibitors for invasion of A549 cells, and the activity of cis-CA seems to be higher than trans-CA. The inhibitory effect of cis-CA on invasion might result from reducing the uPA, MMP-2, MMP-9 activites and the mRNA level of MMP-2 and -9 through inhibiting MAPKs signaling pathways and AP-1 (c-Jun) and NF-B (p65) transcription factors and prohibiting migratory capability of the cells. Biological functions of cis- and trans-cinnamic acid (II) Antituberculosis and synergistic activity on multiple- drug resistant Mycobacterium tuberculosis 　　Tuberculosis (TB) is a contagious disease caused by Mycobacterium tuberculosis. The long course of treatments on TB with a combination of antibiotics leads unfavorable side effects and poor patient compliance which contributes to sustaining multiple-drug resistant tuberculosis (MDR-TB). Cinnamic acid (CA) is a natural occurring phenolic compound with anti-microbial activity. Both trans- and cis-isoforms of CA exist in planta, and cis-cinnamic acid (cis-CA) can be transformed from trans-cinnamic acid (trans-CA) under sunlight. Due to the unavailability of cis-CA, the literature regarding the biological functions of cis-CA is still limited. Although numerous pharmacological effects of trans-CA, such as anti-oxidation, anti-inflammation, and anti-cancer, have been found, cis-CA is considered possesses a higher physiological activity than trans-CA. Uridine diphosphate-glucose pyrophosphorylase (UGPase; EC 2.7.7.9) is the major enzyme that involved to the synthesis of cell wall. Therefore, the development of a new effective synergist or inhibition of UGPase might depress the proliferation of bacteria. The aims of this study were to evaluate the antituberculosis activity of cis-CA and trans-CA and the synergistic effects of these two compounds with two first-line anti-TB antibiotics, isoniazid (INH) and rifampicin (RIF); the involvement of UGPase on the anti-proliferation of multidrug-resistant M. tuberculosis by trans-CA and cis-CA were also determined. Although both of cis-CA and trans-CA decreased the viability of MDR-TB bacilli in a dose-dependent manner, the antituberculosis activity of cis-CA was approximately 120-fold of trans-CA. Furthermore, the cis-CA exhibited higher synergistic effect with INH or RIF against tuberculosis than trans-CA. The micrographs of scanning electron microscope (SEM) display that cis-CA caused an injury on the out-layer of MDR-TB bacilli. The ugp gene fragment was able to obtain from the genome of MDR-TB bacilli by Polymerase Chain Reaction (PCR). By Reverse Transcription Polymerase Chain Reaction (RT-PCR), it was found that the expression of UGPase of the bacilli was decreased after treating with cis-CA and trans-CA for 24 h.In conclusion, both cis-CA and trans-CA can inhibit the proliferation of MDR M. tuberculosis and possess synergistic activity with INH or RIF against tuberculosis; cis-CA is more effective than trans-CA. The mechanism underlying the anti-proliferous activity of cis-CA and trans-CA might be through inhibiting the expression of UGPase to block the cell wall synthesis.

並列關鍵字

cinnamic acid ； matrix metalloproteinase ； human lung cancer cell ； invasion ； metastasis ； tuberculosis ； multiple-drug resistant tuberculosis ； uridine diphosphate-glucose pyrophosphorylase