| 摘要: | 黑色素瘤是一種極惡性的腫瘤，其分化不良，惡性程度高，具高侵襲及轉移性，因此備受重視。在臨床上，黑色素瘤治療方式以手術為主，但若已發生轉移，手術切除一般無法控制病情，此時往往需考慮化學治療、標靶等其他療法，但其毒性及副作用可能使治療成效受限。因此，本研究主要探討在癌症治療上相對較新穎且有潛力的治療方式：誘導癌細胞良性分化及自噬作用。研究發現分化不良的細胞與原發組織相似性低，且其惡性程度較高、侵略性較強，故目前癌症治療策略希望能誘導癌細胞良性分化，研究認為黑色素瘤分化的指標包含：增加黑色素生成、上調tyrosinase活性、增加樹突狀結構產生等。此外，細胞自噬作用過程異常或自噬作用表現低下可能使細胞內清除受損的胞器的能力下降、增加氧化壓力、DNA變異及染色體不穩定性，而與癌症有關。研究亦發現自噬作用可能具抑制癌症的作用，且其作用低下可能與腫瘤形成、進展有關。
十字花科蔬菜具有降低癌症發生的作用，主要和其內富含的indole衍生物indole-3-carbinol (I3C)及其代謝產物3,3'-diindolylmethane (DIM)有關。因此本實驗擬以B16F10黑色素瘤細胞為模式，探討I3C與DIM對黑色素瘤細胞生長、分化及自噬作用的影響。細胞生長實驗部分以MTS及細胞計數法分析I3C及DIM介入對細胞存活率及生長的影響；分化實驗部分觀察細胞典型分化形態，測定細胞內及細胞釋出的黑色素，分析tyrosinase活性，黑色素生合成相關蛋白質MITF、tyrosinase表現，黑色素生合成相關基因MITF、tyrosinase、TRP-1、TRP- 2的mRNA表現，並以穿透式電子顯微鏡觀察黑色素體的形成；自噬作用探討部分分析自噬作用相關蛋白質磷酸化-mTOR、mTOR、LC3-I、LC3-II 之表現，並以穿透式電子顯微鏡觀察自噬體的形成。
結果顯示I3C (50、100 μM)及DIM (10、25、50 μM)處理72小時後，顯著降低細胞數；且於較高劑量I3C (100 μM)及DIM (50 μM)處理後使細胞出現樹突狀結構，於穿透式電子顯微鏡下亦觀察到有較多黑色素體產生，亦顯著增加tyrosinase的活性及胞內及胞外黑色素含量。此外，DIM 50 μM的劑量有增加細胞tyrosinase蛋白質表現量的趨勢並顯著增加tyrosinase、TRP- 2的mRNA表現。另外，I3C及DIM對於自噬作用相關蛋白質磷酸化mTOR (mammalian target of rapamycin)及LC3 (microtubule-associated protein 1 light chain 3)蛋白質表現量無明顯影響，且無觀察到增加自噬體的形成。因此本研究發現I3C及DIM可降低黑色素瘤細胞增殖並誘導其良性分化，但對於黑色素瘤細胞自噬作用無明顯影響。
Melanoma is one of the most malignant tumors due to its poorly differentiated and highly metastasized to vital organs, such as lungs, livers, and brain, so it’s been paid lot of attention. Surgical removal is the main treatment for most melanomas, and also melanoma patients generally undergo chemotherapy, radiotherapy, or other treatments. Although current cancer therapies are significant, these therapies are often with side effects, so that limited their clinical efficacy. Therefore, this study focuses on the relatively novel and potential therapeutic target for cancer: induction of cancer cell differentiation and autophagy. Studies indicate that differentiated melanoma tend to be less aggressive than undifferentiated melanoma, and is associated with slower cell proliferation. The dendritic cell morphology, increased melanin production, increased tyrosinase activity, are markers of differentiated melanoma cells. Some studies indicated autophagy is a mechanism of tumor suppression. Autophagy defect is associated with DNA damage, mutation, and genomic instability, and that contributes to pathological conditions, such as cancer.
It has been indicated that increasing consumption of cruciferous vegetable lowers the risks of cancers, and the glucosinolate derivatives, indole-3-carbinol (I3C) and its metabolite DIM (3,3'-diindolylmethane), are thought to contribute to the anti-cancer activities of these vegetables. Studies have been indicated that I3C can inhibit the growth of various cancer cell lines. However, the effects of DIM on melanoma are not known. Previously, we have indicated that I3C could increase melanin production in melanoma cells, and the biological activities may be associated with DIM. In this study, we investigated the effect of I3C and DIM on cell growth, differentiation and autophagy by using murine B16F10 melanoma cells. The suppressed effect in cell numbers was observed in B16F10 melanoma cells treated with 50-100 μM I3C and 10-50 μM DIM for 48 and 72 hours. To understand the effects of I3C and DIM on cell differentiation, cell morphology will be observed under a microscope, we found that DIM and I3C can induce dendrite morphological changes of B16F10 cells. Furthermore, the content of melanin (intra-cellular and extra-cellular melanin) in B16F10 cells were increased, when treated with 100 μM I3C and 50 μM DIM to the cells. On the other hand, DIM markedly stimulates melanin biosynthesis which was associated with increased tyrosinase activity, gene expression of tyrosinase as well as protein expression of tyrosinase and microphthalmia melanocyte-associated factor (MITF). Finally, we also found 100 μM I3C and 50 μM DIM treated B16F10 cells significantly increased the mature melanosomes compared with the control cells (observations from TEM). However, I3C and DIM treatment didn’t induce the increasing of autophagy related protein expression and autophagosomes after treatment.
In summary, I3C and DIM inhibited cell growth, stimulated expression of markers of melanoma cell differentiation, but showed no effected on autophagy. |
