植物倍半萜類化合物Deoxyelephantopin及其衍生物對小鼠中黑色素瘤肺轉移抑制功效的研究

Abstract

黑色素瘤為皮膚癌中最為嚴重的一種類型，且每年皆具有極高的致病率和死亡率。在臨床上，轉移性的黑色素瘤仍然無法有效治療，且病人在治療後復發率仍然相當高。因此，開發針對轉移性黑色素瘤的新穎療法或預防療法為非常重要的議題。我們實驗室之前發現一種植物倍半萜類化合物deoxyelephantopin (DET) 和其新穎衍生物DETD-35具有抑制人類BRAF(V600E)突變A375異種腫瘤在小鼠體內生長的效果，因此本篇研究主要在探討DET和DETD-35是否對於黑色素瘤肺轉移的體內或體外實驗具有生物活性，試圖提升這兩個植物化合物在治療黑色素瘤的價值。我們實驗室先前建立一株含有hEF1a-elF4g啟動子驅動的冷光素酶報導基因的A375IF4g/Luc細胞株，此細胞經由五次的體外初級培養與反覆尾靜脈注入動物造成高度肺轉移特徵，本論文採取此A375LM5IF4g/Luc細胞株為此研究主要標的試驗細胞。研究結果顯示DET (6 M)和DETD-35 (3 M)可以抑制50% A375LM5IF4g/Luc細胞的生存力。此外，A375LM5IF4g/Luc細胞在24小時和48小時的DET或 DTD-35處理後，發現細胞內有產生空泡的情況。DET和 DTD-35並具有抑制A375LM5IF4g/Luc細胞移動、遷徙和產生群落的能力。西方墨點分析顯示，DET和 DETD-35可以抑制癌細胞轉移相關蛋白質N-cadherin、 vimentin、A-SMA、MMP2、integrin a4 和FAK在A375LM5IF4g/Luc細胞中的表現。此研究並以PLX4032 (一種 BRAFV600E 抑制劑)來當參考抗癌藥一起進行功效分析。流式細胞儀結果顯示PLX4032可以誘導A375LM5IF4g/Luc的細胞週期停留在G1間期，但DET和DETD-35則可以誘導A375LM5IF4g/Luc的細胞週期停留在G2/M間期。而A375LM5IF4g/Luc細胞在DET和 DTD-35處理48小時均有細胞凋亡的產生。在以腹腔注射藥物之NOD/SCID小鼠動物實驗結果顯示，DET (20毫克/公斤) 和 DETD-35 (20毫克/公斤) 可以有效抑制小鼠體內黑色素瘤肺轉移分別降低78% 和75% 腫瘤數目，而PLX4032腹腔注射僅能降低小鼠肺轉移腫瘤24%，但另一批次口服藥物動物實驗顯示可以達到抑制68%的效果。小鼠肺組織經由染色分析後顯示DET (20毫克/公斤) 和 DETD-35 (20毫克/公斤)可以有效抑制下述特徵蛋白質，包括黑色素瘤Mel-A、 細胞增生相關Ki67及血管新生相關VEGF蛋白質的表現，並會誘導細胞凋亡特徵蛋白質cleaved caspase-3 的表達。 總而言之，此篇研究顯示植物倍半萜類化合物deoxyelephantopin (DET) 和其衍生物DETD-35未來具有作為轉移性BRAFV600E突變黑色素瘤治療藥劑的潛力。

Melanoma is a serious form of skin cancer which has high incidence and mortality rate every year worldwide. Currently, metastatic melanoma is still incurable, and the recurrence rate of melanoma after therapy in patients is high. Thus, development of new therapeutic or preventive agents for metastatic melanoma becomes very important. Our lab previously demonstrated that a plant sesquiterpene lactone deoxyelephantopin (DET) and its novel derivative, DETD-35 suppress human orthotopic A375 BRAFV600E mutant melanoma growth in xenograft mice. This study aimed to investigate the bioefficacy of DET and DETD-35 against lung metastasis of melanoma in vitro and in vivo. A lung-seeking metastatic A375LM5IF4g/Luc melanoma cell line, carrying hEF1-elF4g promoter-driven luciferase reporter gene was in-house established through collaboration by five repeated cycles of primary all cultivation of the cells isolated from metastatic cells in lung of NOD/SCID mice. DET and DETD-35 inhibited A375LM5IF4g/Luc melanoma cell viability at IC50 values of 6 M and 3 M, respectively. DET and DETD-35 induced the formation of vacuoles in A375LM5IF4g/Luc melanoma cells at 24 h and 48 h treatments. Both DET and DETD-35 also inhibited migration, invasion, and colony formation of A375LM5IF4g/Luc melanoma cells. DET and DETD-35 were observed to suppress metastatic markers expression, including N-cadherin, E-cadherin, vimentin, SMA, MMP2, integrin 4 and FAK in the melanoma cells. A BRAFV600E inhibitor PLX4032 was used as a reference drug in this study. Flow cytometry analysis revealed that PLX4032 treatment induced G1 phase arrest, while DET and DETD-35 treatment induced G2/M phase arrest in the A375LM5IF4g/Luc cells. Further, DETD-35 and DET treatment induced A375LM5IF4g/Luc cell apoptosis, up to 27% and 32%, respectively at 48 h treatment, while PLX4032 treatment only induced 6% apoptotic cells of the cells. In vivo bioefficacy study using metastatic A375LM5IF4g/Luc melanoma model in NOD/SCID mice showed that both DET (20 mg/kg) and DETD-35 (20 mg/kg) can significantly decrease the numbers of lung tumor foci by 78% and 75%, respectively, with superior effect than the PLX4032 treatment (50 mg/kg) which decreased 24% of lung tumor foci by i.p. intraperitoneally injection. By oral fed drug PLX4032, 68% metastatic melanoma in mouse lung decreased. Overall, the study indicates that phyto-sesquiterpene lactones DET and DETD-35 may be useful in the intervention of lung metastasis of BRAFV600E mutant melanoma.