探討吲哚化合物對大腸直腸癌細胞的腫瘤抑制機轉

在台灣，隨著飲食西化，大腸直腸癌發生率隨之增加，101 年資料顯示大腸直腸癌死亡率位居十大癌症死因第三位。近幾年有相當多的研究顯示，蔬果中所含有之化學物質可以減少癌症的發生率，如富含於十字花科類蔬菜中的吲哚化合物 Indole-3-carbinol(I3C)及其代謝產物 3’3-diindolylmethane (DIM)，已被證實具有抗癌效果。其中在大腸直腸癌細胞株 HT-29 細胞及其他癌症細胞如鼻咽癌細胞株CNE-2 中能調控細胞週期及細胞凋亡相關蛋白表現變化抑制癌細胞。但在 DLD1 及 Hct15 這兩株大腸直腸癌細胞株的詳細機轉卻尚未被釐清，因此本篇研究將探討吲哚化合物 I3C 及 DIM 對於 DLD1及 Hct15 細胞株的影響及詳細作用機轉為何。 我們利用細胞計數(cell count)和細胞存活率分析 3-(4,5-Dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT assay)偵測吲哚化合物對於癌細胞的細胞毒性。首先先將 DLD1 及Hct15 細胞株分別處理不同濃度的吲哚化合物(DMSO、DIM 10 μM、DIM 50μM、I3C 100μM)，0、24、48、72 小時後觀察其細胞數量變化。結果顯示， Hct15 及 DLD1 這兩株細胞皆會隨著處理劑量及時間的增加而減少細胞存活率，且 DIM 效果較 I3C 為顯著。進一步利用 hoechst 33342 染色也看到了在 DLD1 及 Hct15 細胞中經過 DIM 50μM 處理 48 小時後均會造成染色質濃縮，再利用西方墨點法(Western Blot)來偵測吲哚化合物調控細胞蛋白的表現，藉此了解吲哚化合物是透過何種路徑來抑制癌細胞生長。發現 DLD1 細胞經由 DIM 50μM 處理 24 小時後，會促使細胞凋亡相關蛋白 Bad 表現量上升及抗凋亡蛋白 Bcl2 表現量下降，但在 Hct15 細胞中 Bad及 Bcl2 表現量皆下降，而 Hct15 及 DLD1 細胞經 DIM50 μM 處理後其下游凋亡相關蛋白 caspase3 及 PARP 也都沒有剪切現象發生。除此之外，腫瘤抑制蛋白 P53 以及細胞自噬相關蛋白的 LC3II在經過 DIM 50 μM 處理後表現均有明顯上升。 綜合以上結果，可得知透過吲哚化合物確實會抑制大腸直腸癌細胞 DLD1 和 Hct15 細胞生長，達到抗癌效果，但其詳細路徑可能分別與細胞凋亡，細胞自噬與細胞週期調控有關，未來仍需進一步研究其詳細機轉。

In Taiwan, along with economic development and westernized diet, increased incidence of colorectal cancer, 101 -year data show that the mortality rate for colorectal cancer ranks third in the top ten causes of death of cancer, recently, a considerable number of studies have shown that chemical substances contained in fruits and vegetables can reduce the incidence of cancer, such as Indole compounds, are rich in cruciferous vegetables, have been reported to have anti-cancer effect. Indole-3-carbinol (I3C) and its derivative 3’3-diindolylmethane (DIM), were confirmed to suppress cell growth through the regulation of cell cycle and apoptosis related protein in colon cancer cell HT-29 and another cancer cells. However, its detailed molecular mechanism is unclear both in DLD1 and Hct15 colon cancer cell lines. In this study, we want to understand the tumor suppressed mechanism of Indole-3-carbinol (I3C) and 3’3-diindolylmethane (DIM) in DLD1 and Hct15 colon cancer cell lines. We used cell count and MTT assay to detect the cytotoxicity of these indole compounds. DLD1 and Hct15 cell lines were treated with different dose of indole compounds (DIM10μM, DIM50μM, I3C100μM) at different time (0, 24, 48, 72 hours), then the cell number and viability were observed.Our result showed that cell viability of DLD1 and Hct15 cell lines were significant decrease after treated with indole compounds in dose-dependent and time-dependent manner, and DIM was more effectively than I3C. Furthermore, we further use Hoechst 33342 staining to detect DNA condensation of DLD1 and Hct15 cell lines. Result show that both of DLD1 and Hct15 cell lines have DNA condensation after treated with DIM50μM for 48 hours. We also used Western blot detected the expression of cell death related protein and understand the pathway of cell growth inhibition. Our data show that after treated with DIM 50μM for 24 hours, Bad, the apoptosis-related protein expression increased and anti-apoptotic protein Bcl2 expression decreased in DLD1, but not in Hct15. On the other hand, data didn’t show any cleavage of caspase-3 and PARP in both of two cell lines after treated with DIM 50μM. In addition, the tumor suppressor protein P53 and autophagy-related proteins LC3II expression were significant increased after treated with DIM 50μM. In summary,we demonstrate that indole compounds have anti-cancer effect, can inhibit cell growth through in DLD1 and Hct15 colon cancer cells. However, its detailed molecular mechanism is uncleared.We will continue to investigative the cell death type and pathway of cancer cells in the future.