EF-24抑制人類子宮頸癌細胞轉移和侵襲之機制探討

根據衛生福利部的統計，民國106年子宮頸癌在台灣女性癌症排名第七。造成子宮頸癌病患死亡的最大原因是癌症的轉移 (metastasis)，在癌症轉移中，癌細胞會釋放出趨化因子、生長因子以及基質金屬蛋白水解酶 (matrix metalloproteinases, MMPs)，分解細胞外基質 (extracellular matrix, ECM)，形成血管新生，並藉由血液流動轉移到其他組織。Diphenyldifluoroketone (EF-24)是一種人工合成的薑黃類似物，在先前的文獻中已經證實，EF-24可以抑制黑色素瘤細胞和卵巢癌細胞的遷移，在膽管癌細胞的侵襲中也有有效的抑制作用。但EF-24在抑制子宮頸癌的遷移和侵襲中的作用和機制仍然是未知的。因此本篇研究將探討EF-24在抑制子宮頸癌轉移的詳細機制。12-O-tetradecanoyl-phorbol-13-acetate (TPA)也稱為PMA，是一種腫瘤促進因子，有研究指出TPA會促進細胞分泌MMP-9，使細胞遷移的能力上升。因此在本實驗中將會使用TPA作為誘發因子並同時以EF-24來處理子宮頸癌細胞株 (HeLa, SiHa cell lines)。首先利用MTT assay分析結果顯示EF-24以及TPA不會影響子宮頸癌細胞的存活率。接著以wound healing assay、migration assay以及invasion assay分析發現EF-24可以明顯抑制TPA所誘導的細胞遷移和侵襲能力。再來利用gelatin zymography的方式分析細胞分泌出的MMP-9，結果發現EF-24能抑制TPA所誘發的MMP-9活性。下一步利用RT-PCR以及real-time PCR的方式進行分析，結果發現EF-24能降低TPA所誘導的MMP-9 mRNA表現，在MMP-9蛋白表現的分析中也得到了相同的結果。在另一方面，EF-24能有效抑制p38的磷酸化。總結以上結果，在子宮頸癌細胞中，EF-24可能透過抑制TPA所誘發的p38的磷酸化，抑制細胞分泌MMP-9，使癌細胞遷移和侵襲能力降低，但是其上游訊息傳遞路徑有待更進一步的實驗來確認。

According to Ministry of Health and Welfare, cervical cancer ranked seventh in cancer mortality of women in 2017. The main cause of death in cervical cancer is metastasis. During cancer metastasis, the extracellular matrix (ECM) was decomposed by cancer-released chemokines, growth factors and matrix metalloproteinase (MMPs), and promote cancer angiogenesis and distal metastasis through the blood flow. Diphenyl difluroketone (EF-24), a curcumin analog, has been indicated that EF-24 could inhibit migration of melanoma cell and ovarian cancer cell. EF-24 could also effectively inhibit invasion of cholangiocarcinoma. However, the anti-cancer effect of EF-24 exerts on cervical cancer cells remains unclear. The present study aimed to examine the anti-metastatic properties of EF-24 in cervical cancer cell lines. 12-O-tetradecanoylphorbol-13-acetate (TPA), also called PMA, is a tumor promoter and may induce MMP-9 expression in tumor cell that could increase the metastatic ability. In this study, HeLa cells and SiHa cells were treated with TPA and combined with or without EF-24. Cell viability was examined by MTT assay. Result shows that EF-24 and TPA treatment will not affect cervical cancer viability. The results of wound healing assay and boyden chamber assay showed that EF-24 could suppress TPA-induced cervical cancer migration and invasion. We also found that EF-24 inhibits TPA-induced MMP-9 enzyme activity by gelatin zymography assay. Furthermore, results from RT-PCR (Reverse-transcription PCR) and Real-time PCR indicated that EF-24 significantly inhibited TPA-induced MMP-9 mRNA expression. EF-24 could also suppress TPA-induced MMP-9 protein expression. In addition, the results indicated that EF-24 inhibited the phosphorylation of p38. In conclusion, we found EF-24 might inhibit TPA-induced migration and invasion of cervical cancer cell through modulating MMP-9 activity via downregulating p38 pathway, but the detail underlying mechanisms remains to be confirmed.