探討微環境及植化素對大腸直腸癌癌症幹細胞發展之影響

Abstract

國人十大死因之首為癌症，其中大腸結直腸癌(colorectal cancer, CRC)位居第三，與國人之飲食、生活習慣、遺傳等因素相關。過去研究指出微環境改變和癌症幹細胞存在對癌症發生、轉移與再發具有密不可分之關係。本研究目的為探討植化素與微環境調控對大腸直腸癌癌症幹細胞(colorectal cancer stem cells，CCSCs)發展之影響。實驗建立以CRC病患之正常、瘜肉與癌組織分析CCSCs marker (CD133與CD44)之表現量及代謝體，接續建立CRC初代細胞培養平台，觀察植化素對各CRC初代細胞中CCSCs及代謝體之影響，再者以CRC細胞株HCT-116、HT-29、SW480及SW620細胞株中CCSCs表現分析及微環境因子對CCSCs發展之影響，以CCSCs表現量高低，模擬CRC細胞及瘜肉細胞，針對CCSCs高度表現之HT-29細胞株給予植化素curcumin (Cur)、resveratrol (Res)及4-Acetylantroquinonol B (4AAQB)探討其對細胞存活率、凋亡、CCSCs表現的影響，而CCSCs表現量低之SW480細胞株則給予不同微環境因子如reactive oxygen species (ROS)、epidermal growth factor (EGF)及transforming growth factor (TGF)誘導CCSCs表現。實驗結果發現CRC腺瘤性息肉(N=61)的代謝特徵相較於CRC（N = 57）組織，其磷酸腺苷（Adenosine monophosphate, AMP），adenine，5''- methythioadenosine，3-hydroxybutyric acid ，prostaglandin E2， threonine 和 glutamine具統計上顯著差異（P <0.05）。因CRC初代細胞培養平台建置尚未完成，且以微環境因子誘導CRC細胞株表現CCSCs部分，發現以單一因子無法有效誘導CCSCs表現，因此以CRC細胞株進行研究。結果發現以50 μM curcumin處理48小時確實增加CRC及CCSCs的細胞凋亡，但僅在CD44+細胞具顯著性。在CRC細胞株的代謝特徵的研究，curcumin處理後CD44-細胞中glyceraldehyde和hydroxypropionic acid之含量增加，但以curcumin處理的CRC和CD44+細胞則glutamine含量減少。基於我們的人體組織和癌細胞株的代謝概況比較，我們認為，curcumin-CD44+耦合在細胞膜上可能對glutamine的運輸產生某些阻斷作用，抑制其進入細胞內，因此降低了glutamine在CD44+細胞中的含量以至於誘導細胞凋亡的發生。

Colorectal cancer (CRC) is the three major cause of cancer-related mortality in Taiwan. The risk factors of CRC include age, life style and family history. This study investigated the effect of microenvironment and phytochemicals on the development of colorectal cancer stem cells (CCSCs) and its possible mechanism. We analyzed CRC patient’s normal, adenomatous polyps and tumor tissue for expression of CCSCs (CD133 and CD44) and metabolic profiles. We also tried to establish CRC primary cells for investigating the effect of phytochemicals and microenvironmental factors on the CCSCs and metabolic profiles. In addition, the microenviroments factors including reactive oxygen species (ROS)、epidermal growth factor (EGF)及transforming growth factor (TGF) were used to induce the expression of CCSCs. However, both of the attempt of establishing CRC primary culture and induction of CCSCs by microenvironmental factors failed, we decided to use CRC cell lines (HCT-116, HT-29, SW480 and SW620) model to analyze their expression of CCSCs and metabolic profiles. Comparison of the metabolic profiles of human adenomatous polyp (N=61) and colorectal cancer (CRC) (N=57) tissue found statistically significant differences (p<0.05) in their composition of Adenosine monophosphate (AMP), adenine, 5’-methythioadenosine, 3-hydroxybutyric acid, prostaglandin E2, threonine and glutamine. Our cell culture model study found that curcumin treatment (50 μM for 48 h) did increased apoptosis of CRC cells and CCSCs, but only the CD44+ cells reached statistically significant. Further metabolic profile studies of the CRC, CD44+ and CD44- cells indicated that curcumin treatment increased glyceraldehyde and hydroxypropionic acid in CD44- cells, but decreased glutamine content in both curcumin-treated CRC and CD44+ cells. Based on the comparison of the metabolic profiles of human tissues and cancer cells we suggest that curcumin might couple with CD44 and that curcumin-CD44+ coupling at the cell membrane might have some blocking effect on the transport of glutamine into the cells, thus decreasing the glutamine content in the CD44+ cells and inducing apoptosis.