柑橘類果皮中多甲氧基類黃酮抑制前列腺癌細胞生長作用之探討

Inhibitory Effect of Citrus Polymethoxy Flavones (PMFs) on the Growth of Prostate Cancer Cells

趙彥翔(Yen-Hsiang Chao)

指導教授 ： 蘇正元

中原大學/理學院/生物科技研究所/碩士(2018年)

https://doi.org/10.6840/cycu201800708

摘要

民國106年十大死因中，惡性腫瘤高居兩性十大死因第一名，且前列腺癌為男性惡性腫瘤十大死因中第七名，然而傳統的癌症治療技術 (包括手術切除、放射和化學性治療等)，雖能移除癌細胞，但仍有復發或轉移的情況發生。本研究乃探討柑橘果皮內的植化素-多甲氧基類黃酮 (polymethoxyflavones, PMFs) 與其衍生化合物對人類前列腺癌細胞生長抑制作用與相關機制之探討。研究結果顯示，tangeritin的衍生物4',5-didemethyltangeretin (本論文中簡稱PMF1) 能最有效抑制人類前列腺癌 LNCaP 和 PC-3細胞生長 (IC50分別為14.3和13.5 µM) 和非貼附性生長，但對人類正常前列腺 RWPE-1細胞有相對較低的細胞毒性 (IC50為28.0 µM)。PMF1能調控 LNCaP 細胞內細胞凋亡相關蛋白 (提升 Bad，以及降低 Bcl-2、pro-caspase3 和 PARP等) 表現，進而誘導細胞凋亡 (SubG1期增加)。同時 PMF1也能降低 PC-3細胞內細胞週期調控蛋白 CDK1表現量，使細胞生長停滯於 G2/M 期。PMF1亦降低 LNCaP 和 PC-3細胞內表觀遺傳調控蛋白之表現量，包括DNA methyltransferase3b (DNMT3b)、histone deacetylases2 (HDAC2) 和 HDAC4/5/9等，並顯著提升腫瘤抑制基因 p21 mRNA 和蛋白質表現量。甲基化專一性聚合酶鏈反應 (methylation specific PCR, MSP) 結果顯示 PMF1 能降低 LNCaP 細胞內 p21啟動子甲基化程度；另 PMF1亦干擾體外甲基轉移酶 (M.SssI) 活性，並能與 LNCaP 細胞裂解液中 DNMT1、2和3b 相互結合。再者，本研究從 LNCaP 細胞中分離出高表達 CD166的癌類幹細胞 (cancer stem-like cells, CSLCs)，而 PMF1能有效抑制 LNCaP CSLCs 非貼附性生長。綜合以上結果得知，PMF1能透過影響前列腺癌細胞內表觀遺傳調控酵素，進而增加 p21表現，促使前列腺癌 LNCaP 細胞發生細胞凋亡和 PC-3細胞週期停滯。關鍵字: 細胞凋亡、癌類幹細胞、表觀遺傳學、多甲氧基類黃酮、前列腺癌

關鍵字

多甲氧基黃酮類化合物 ； 前列腺癌 ； 表觀遺傳學 ； 細胞凋亡 ； 細胞週期 ； 癌類幹細胞

並列摘要

Prostate cancer is the second leading cause of cancer death in men in the United States. Polymethoxyflavonoids (PMFs), such as tangeretin and nobeliten, which exclusively exist in citrus fruit peels have been demonstrated to exhibit various bioactivities. In this study, we investigated the inhibitory effect of eight derives of tangeretin (PMFs 1-4) and nobelitin (PMFs 5-8) against human prostate cancer LNCaP and PC-3 cells. The results showed that 4’,5-didemethyltangeretin (PMF1) significantly inhibited the growth of LNCaP and PC-3 cells (IC50 14.3 and 13.5 µM, respectively) while PMF1 at 15 µM didn’t affect the growth of human normal prostate RWPE-1 cells. PMF1 also suppressed the anchorage-independent growth of LNCaP and PC-3 cells. PMF1 increased Bad expression and decreased procaspases3, pro-PARP, and Bcl-2 expressions, there by inducing apoptosis in LNCaP cells. PMF1 downregulated DNA methyltransferase (DNMT) 3b and histone deacetylases (HDACs) 1, 2, and 4/5/9 in LNCaP cells. PMF1 also induced cell cycle arrest in PC-3 cells in which PMF1 decreased protein expressions of CDK1, DNMTs 1 and 3b, and HDACs 2 and 4/5/9. Furthermore, PMF1 increased tumor suppressor gene p21 mRNA and protein levels in both LNCaP and PC-3 cells. Methylation-specific PCR results revealed that PMF1 decreased methylated p21 promoter level in LNCaP cells. We further demonstrated that PMF1 supressed DNA methyltransferase (M.SssI) activity in vitro, PMF1 had ability to bind to DNMTs 1, 2 and 3a ex vivo in LNCaP cells. Additionally, PMF1 inhibited the anchorage-independent growth of isolated LNCaP cancer stem-like cells (CSLCs) with high CD166 mRNA expression. These results suggested that PMF1 might effectively inhibit the growth of human prostate cancer cells through epigenetic upregulation of p21 pathway and cancer stem cells.
Key words : apoptosis, cancer stem-like cells, cell cycle, epigenetics, polymethoxyflavones (PMFs), prostate cancer

並列關鍵字

Polymethoxyflavones (PMFs) ； prostate cancer ； epigenetics ； apoptosis ； cell cycle ； cancer stem-like cells

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