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標題: 26-methoxycholesta-1,4-dien-3-one 抑制黑色素瘤細胞存活的活性分析
Activity Analysis of 26-Methoxycholesta-1,4-dien-3-one for Inhibiting the Survival of Melanoma Cells
作者: 洪琪雅
Chi-Ya Hung
關鍵字: 類固醇;馬紅球菌;黑色素瘤細胞;steroid;Rhodococcus equi;melanoma cell
類固醇化合物廣泛存在於自然界的生物體內,並扮演調節生理功能的重要角色。在醫療上,常用於抗發炎或免疫調節的相關疾病,甚至做為抗癌化療時的輔助藥物。馬紅球菌 (Rhodococcus equi) 是一株具有代謝固醇能力的革蘭氏陽性菌,而其代謝路徑的中間產物,1,4-雄二烯二酮 (1,4-androstadiene-3,17-dione, ADD) 是生產固醇類藥物的重要前驅物。過去本實驗室已經將 R. equi 中降解 ADD 的關鍵酵素之鐵氧蛋白還原酶 (kshB) 進行基因剔除,再經過紫外光誘導突變而得一株能大量累積 ADD 的突變株 R. equi UV1。在實驗中也發現 UV1 菌株的發酵代謝物中出現類固醇代謝物 (26-hydroxycholesta-1,4-dien-3-one, HCD),並發現該代謝物有癌細胞毒殺能力。為了進一步使該固醇類化合物具有抗癌藥物的潛力,增加其穩定性及延緩 HCD 在生物體內的降解時間。實驗室已將此類固醇進行甲基化,得到 26-methoxycholesta-1,4-dien-3-one (mHCD)。本研究目的是為了解甲基化後得到的 mHCD 產物,對於不同種癌細胞是否具有抑制潛力。利用細胞毒性測試 (MTT assay) 結果發現, mHCD 對於惡性黑色素瘤細胞 (A375)、乳腺癌細胞 (MDA-MB-231、MCF-7)、肺癌細胞 (A549) 和肝癌細胞 (HepG2) 皆有抑制效果。而其中又以 A375 的抑制效果最為顯著,其 IC50 為 10.61 µM,且對控制組之正常皮膚纖維細胞 (CCD966-SK) 在最高劑量 20 µg/ml 沒有明顯的抑制效果。且相較於甲基化前的 HCD 化合物增加約 6.5 倍的抑制效果。表明 mHCD 可以有效使 A375 細胞造成細胞凋亡,抑制癌細胞的生長與複製。
另外用 Annexin V-FITC/PI 分析 mHCD 對 A375 及 CCD966-SK 細胞是否造成細胞凋亡,發現 mHCD 會使 A375 細胞導向細胞凋亡的途徑,且對正常細胞 CCD966-SK 不會有影響。因此,mHCD 可以做為抑制黑色素皮膚癌的潛力抗癌藥物。

Steroids, present in many natural organisms, play an important role in regulating a variety of physiological functions. For the medical uses, they have been used for anti-inflammatory or immune-related diseases as well as an adjuvant in anticancer chemotherapy. Rhodococcus equi, a gram-positive bacterium, has the ability to metabolize sterols. Of the intermediates in the sterol degradation pathway, 1,4-androstadiene-3,17-dione (ADD) has been widely used as a precursor for the production of many steroid drugs. In previous studies, the gene encoding the key component of ADD-degrading enzyme of R. equi, kshB, was deleted and the knockout strain was further mutated by UV mutagenesis. A high ADD production mutant, named R. equi UV1, was then identified. Besides ADD, UV1 also produces 26-hydroxycholesta-1,4-dien-3-one (HCD) that was found able to inhibit the growth of cancer cells. In order to delay the bio-degradation of HCD, this compound was methylated at the C26 hydroxyl group in the previous experiment. The purpose of this study is to determine the inhibitory effect of 26-methoxycholesta-1,4-dien-3-one (mHCD) on various types of cancer cells. Based on MTT assay, mHCD has an inhibitory effect on the growth of human malignant melanoma cell line A375, human mammary gland/breast cell line MCF-7 and MDA-MB-231, human lung carcinoma cell line A549, and human hepatoblastoma cell line HepG2. Among these cancer cells, A375 was the most vulnerable one with an EC50 of 10.61 µM. By contrast, human skin fibroblast normal cell line CCD966-SK wasn't inhibited by 20 µg/ml mHCD. The result also indicates that the mHCD increase the inhibitory effect by a factor of 6.5. Therefore, we suspect that mHCD may cause an apoptosis in A375 but not CCD966-SK. Additionally, Annexin V-FITC/PI was used to analyze whether mHCD caused apoptosis in A375 and CCD966-SK cells. It was found that mHCD led to A375 cells apoptosis but wasn't in normal cells CCD966-SK. Therefore, mHCD can be used as an anticancer drug for skin cancer inhibition.
Rights: 同意授權瀏覽/列印電子全文服務,2018-07-30起公開。
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