攝護腺癌是世界排名第5的常見癌症。超過80%的攝護腺癌症病患死於骨轉移。咖啡酸苯乙脂(caffeic acid phenethyl ester, CAPE)為蜂膠萃取物中主要具生物活性的一種成分。我們利用wound healing和transwell的實驗證明咖啡酸苯乙脂可以抑制PC-3和DU-145攝護腺癌細胞的移動與侵略性，並透過gelatin zymography證明咖啡酸苯乙脂可抑制MMP-9的活性。PC-3攝護腺癌細胞經咖啡酸苯乙脂處理後，利用gene array和RT-qPCR發現Wnt訊號傳遞中的抑制劑ROR2和KREMEN1的基因表現量上升，但HRAS, RAF1, PI3KR1, AKT1, AKT2, GSK3A和EGF的基因表現量則受到抑制。利用高通量西方點墨分析系統(high-throughput Micro-Western Array, MWA)分析結果發現經咖啡酸苯乙脂處理後，non-canonical Wnt的ROR2蛋白質表現上升，但-catenin、NF-B活性、PI3K-Akt和epithelial-mesenchymal transition (EMT)表現量則受到抑制。我們利用Wnt的TCF-LEF promoter接上冷光蛋白質基因來驗證經咖啡酸苯乙脂處理後會抑制攝護腺癌細胞的Wnt訊號傳遞。利用腹腔注射將咖啡酸苯乙脂分別給予經尾靜脈或後腿注射接種PC-3攝護腺癌細胞後的裸鼠身上，可以減少癌細胞的轉移與增生。免疫組織染色證明帶有PC-3癌細胞的裸鼠經過咖啡酸苯乙脂治療後，ROR2的蛋白質表現量上升，但Ki-67、Frizzled 4、NF-B p65、 MMP-9、 Snail、 β-catenin和磷酸化IκBα的蛋白質表現量則下降。臨床的證據指出，HRAS、RAF1、AKT2、GSK3A、EGF、nuclear β-catenin、NF-B p65、MMP9和snail在具轉移的前列腺癌細胞的表現量較高且預後較差，而KREMEN1、IκBα、MKP-3和BMP4的表現量則較低。根據我們臨床上以及細胞實驗分析結果，ROR2的表現量在攝護腺癌細胞中較低，特別是轉移的癌細胞。在DU-145和PC-3細胞中大量表現ROR2蛋白質明顯地提高E-cadherin表現量，但抑制vimentin、MMP-9，和細胞核內β-catenin的蛋白質量。利用transwell和wound healing assay證明在DU-145和PC-3攝護腺癌細胞中的過度表現ROR2蛋白質會抑制細胞的移動力。利用MWA分析發現在PC-3細胞中過度表現ROR2會導致PIAS3蛋白質量上升，但會抑制Stat3、NF-κB、phospho-Akt Ser473和Thr308。在PC-3攝護腺癌細胞中降低PIAS3表現量會增強細胞移動力和提高phospho-Akt Thr308及MMP-9的蛋白質量，但這些影響會因大量表現ROR2而被廢除。我們的研究結果推測ROR2很可能在攝護腺癌細胞中扮演一個腫瘤抑制蛋白質的角色，且咖啡酸苯乙脂調控的基因與蛋白質表現在臨床上對攝護腺癌細胞的轉移與預後是具有影響力的，我們推斷咖啡酸苯乙脂對癌症病人的治療是有幫助的。

Prostate cancer (PCa) was the fifth most common cancer overall in the world. More than 80% of patients died from PCa developed bone metastases. Caffeic acid phenethyl ester (CAPE) is a main bioactive component of honeybee hive propolis. We demonstrated that CAPE treatment suppressed the migration and invasion of PC-3 and DU-145 prostate cancer (PCa) cells as well as MMP-9 activity as determined by wound healing, transwell, and gelatin zymography assays. CAPE treatment elevated gene abundances of Wnt signaling inhibitor receptor tyrosine kinase-like orphan receptor 2 (ROR2) and KREMEN1, but repressed gene abundances of HRAS, RAF1, PIK3R1, AKT1, AKT2, GSK3A and EGF in PC-3 PCa cells as determined by gene array Gene Set Enrichment Analysis (GSEA) analysis and qRT-PCR assay. Analysis using Micro-Western Array (MWA), a high-throughput antibody-based proteomics platform indicated that CAPE treatment induced ROR2 in non-canonical Wnt signaling pathway but suppressed abundance of -catenin, NF-B activity, PI3K-Akt signaling, and epithelial-mesenchymal transition (EMT). TCF-LEF promoter binding assay confirmed that CAPE treatment reduced canonical Wnt signaling. Intraperitoneal injection of CAPE reduced the metastasis and proliferation of PC-3 xenografts in tail vein and hind leg injection nude mice model, respectively. Immunohistochemical staining demonstrated that CAPE treatment increased abundance of ROR2 and Wnt5a but decreased protein expression of Ki67, Frizzle 4, NF-B p65, MMP9, Snail, β-catenin, and phosphorylation of IκBα. Clinical evidences revealed higher expression level of HRAS, RAF1, AKT2, GSK3A, EGF, nuclear β-catenin, NF-B p65, MMP9 and snail, as well as lower expression level of KREMEN1, IκBα, MKP-3, and BMP4 positive correlation with aggressive diseases and poor outcomes. Over-expression of ROR2 in DU-145 and PC-3 cells suppressed migration and invasion as determined with transwell and wound healing assay. Over-expression of ROR2 in DU-145 and PC-3 PCa cells significantly elevated E-cadherin, and suppressed vimentin, MMP-9, as well as nuclear β-catenin in protein level. MWA revealed that over-expression of ROR2 led to elevate protein level of PIAS3, but suppressed abundance of Stat3, NF-κB, phosphor-Akt Ser473 and Thr308 in PC-3 cells. Knockdown PIAS3 in PC-3 PCa cells enhanced cell migration and elevated phospho-Akt Thr308 and MMP-9, but these effects were abolished in ROR2 over-expressed PC-3 cells. Our study suggests that CAPE treatment might benefit prostate cancer patients with aberrant elevation of canonical Wnt and EGFR signaling and ROR2 is a novel tumor suppressor in PCa cells.

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