Bornyl cis-4-Hydroxycinnamate造成黑色素癌細胞凋亡及粒線體失活與內質網壓力之探討

The Effect of Bornyl cis-4-Hydroxycinnamate on Melanoma Cell Apoptosis Is Associated with Mitochondrial Dysfunction and Endoplasmic Reticulum Stress

楊子姸(Tzu-Yen Yang)

指導教授 ： 吳美莉 ； 吳裕仁

國立屏東科技大學/農學院/食品科學系所/博士(2020年)

https://doi.org/10.6346/NPUST202000418

摘要

從荖葉莖中所分離出的活性化合物Bornyl cis-4-hydroxycinnamate，具有用作抗癌藥的潛力。在這項研究中，研究了Bornyl cis-4-hydroxycinnamate對黑素瘤細胞增殖，蛋白質表達以及細胞遷移和侵襲的影響。透過使用流式細胞儀分析了由Bornyl cis-4-hydroxycinnamate誘導的黑色素瘤細胞系凋亡的早期階段，並採用了比較蛋白質質體學技術研究了該化合物對A375細胞蛋白質表現的影響。 A375細胞的二維電泳（2-DE）分析顯示35種蛋白質的表現水平發生了顯著變化，其中18種蛋白質表現量上升而17種蛋白質表現量下降。蛋白質體學研究確定了幾種與內質網壓力（ER Stress）和粒線體功能失活有關的蛋白質。該處理還導致粒線體膜電位顯著下降，細胞色素C釋放到細胞質中，激活Bax、Bad、caspase-3、caspase-9表現量，以及造成p-Bad、Bcl-2，Bcl-xl和Mcl-1的表現量降低，由結果顯示 Bornyl cis-4-hydroxycinnamate誘導的凋亡是由粒線體誘導的caspase依賴性途徑所造成。Bornyl cis-4-hydroxycinnamate所引起的的細胞死亡也會經由PERK-eIF2α-ATF4-CHOP信號傳遞被活化。我們的研究結果支持Bornyl cis-4-hydroxycinnamate誘導的黑色素瘤細胞凋亡與caspase活化，粒線體功能失活和內質網壓力相關的機制有相關聯性。
另外，本文也研究Bornyl cis-4-hydroxycinnamate（1–6 µM）處理過的黑色素瘤細胞的細胞遷移和侵襲的研究。為了驗證Bornyl cis-4-hydroxycinnamate是否對黑色素細胞瘤具有潛在的抗轉移作用，在研究中使用Boyden測定法和Transwell對黑色素細胞瘤進行了細胞遷移和侵襲測定。用明膠酶譜法測定MMP-2和MMP-9的酵素活性。並進行MMP-2，MMP-9和TIMP-1 / 2以及MAPK，FAK / PI3K / Akt / mTOR信號傳遞中的關鍵分子的蛋白質免以色分析法。由結果可以發現，Bornyl cis-4-hydroxycinnamate是潛在有用的藥物，通過抑制FAK / PI3K / Akt / mTOR和MAPKs降低MMP-2和MMP-9的表達，從而抑制黑素瘤細胞的遷移和侵襲並改變黑素瘤細胞的轉移。這些研究結果發現Bornyl cis-4-hydroxycinnamate具有作為化學治療劑的潛力，並且值得進一步研究以用於黑色素細胞瘤的治療。
關鍵詞：黑色素瘤、Bornyl cis-4-hydroxycinnamate、蛋白質體學、粒線體功能失活、細胞凋亡、內質網壓力，細胞侵襲、細胞遷移

關鍵字

黑色素瘤 ； Bornyl cis-4-hydroxycinnamate ； 蛋白質體學 ； 粒線體功能失活 ； 細胞凋亡 ； 內質網壓力 ； 細胞侵襲 ； 細胞遷移

並列摘要

Bornyl cis-4-hydroxycinnamate, an active compound isolated from Piper betle stems, has the potential for use as an anti-cancer agent. In the study were investigated the effects of bornyl cis-4-hydroxycinnamate on melanoma cell proliferation, protein expression and cell migration and invasion in melanoma cells. The flow cytometric analysis to examine the early stages of apoptosis induced by bornyl cis-4-hydroxycinnamate in melanoma cell lines and employed comparative proteomic analysis to investigate the effects of bornyl cis-4-hydroxycinnamate on protein expression in A375 cells. Two-dimensional electrophoresis (2-DE) analysis of A375 cells showed that the expression levels of 35 proteins were significantly altered, with 18 proteins upregulated and 17 downregulated. The proteomics study identified several proteins that are involved in endoplasmic reticulum stress (ER stress) and mitochondrial dysfunction. The treatment also resulted in a marked decline of the mitochondrial membrane potential, in cytochrome C release into the cytosol, in the activation of Bax, Bad, caspase-3, and caspase-9, and in the decreased expression of p-Bad, Bcl-2, Bcl-xl, and Mcl-1, indicating that apoptosis induced by bornyl cis-4-hydroxycinnamate was mediated by the mitochondria through the caspase-dependent pathway. Bornyl cis-4-hydroxycinnamate-related cell death also implied that the PERK)–eIF2α–ATF4–CHOP signal pathways was activated upon bornyl cis-4-hydroxycinnamate treatment. Altogether, our results support that bornyl cis-4-hydroxycinnamate-induced apoptosis in melanoma cells is associated with mechanisms correlated with the activation of caspase cascades, mitochondrial dysfunction, and endoplasmic reticulum stress, and indicate that this molecule has the potential to be developed as a chemotherapeutic agent for human melanoma. Cell migration and invasion were compared on melanoma cell lines treated bornyl cis-4-hydroxycinnamate (1–6 µM). To examine whether bornyl cis-4-hydroxycinnamate has a potential anti-metastatic effect on melanoma cells, cell migration and invasion assays were performed using a Boyden chamber assay and a transwell chamber on melanoma cell. Gelatin zymography was employed to determine the enzyme activities of MMP-2 and MMP-9. Cell lysates were collected for western blotting analysis of MMP-2, MMP-9 and TIMP-1/2, as well as key molecules in the MAPKs, FAK/PI3K/Akt/mTOR signaling pathways. Our results demonstrated that bornyl cis-4-hydroxycinnamate is a potentially useful agent that inhibits melanoma cell migration and invasion and alters melanoma cell metastasis by reducing MMP-2 and MMP-9 expressions through inhibition of the FAK/PI3K/Akt/mTOR and MAPKs signaling pathways. These findings suggested that bornyl cis-4-hydroxycinnamate has potential as a chemotherapeutic agent and warrants further investigation for use in the management of human melanoma.

Keywords: melanoma; bornyl cis-4-hydroxycinnamate; proteomic; mitochondrial dysfunction; apoptosis; endoplasmic reticulum stress, Cell invasion; cell migration

並列關鍵字

melanoma ； bornyl cis-4-hydroxycinnamate ； proteomic ； mitochondrial dysfunction ； apoptosis ； endoplasmic reticulum stress ； Cell invasion ； cell migration

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