大蒜精油對人類皮膚黑色素細胞癌之抑制活性與機制探討

Investigation of anticancer activity and mechanism of garlic essential oil against human melanoma cells

包容(Jung Pao)

指導教授 ： 沈立言

國立臺灣大學/生物資源暨農學院/食品科技研究所/碩士(2011年)

https://doi.org/10.6342/NTU.2011.10456

摘要

皮膚癌是全球罹患率最高的癌症之一，可分為非黑色素細胞癌 (non-melanoma skin cancer, NMSC)與黑色素細胞癌(melanoma)，而黑色素細胞癌(melanoma)是皮膚癌中最惡性的一種，容易轉移至肝、肺及腦，亦會抵抗化學療法及放射線療法，致死率較高。大蒜精油活性成分-二丙烯基硫化物（allyl sulfides）以二丙烯基單硫化物（diallyl sulfide, DAS）、二丙烯基二硫化物（diallyl disulfide, DADS）與二丙烯基三硫化物（diallyl trisulfide, DATS）對多種癌細胞株具有抑制活性，但目前大蒜精油在人類皮膚癌細胞的研究卻很少見。因此，本研究以水蒸氣蒸餾法萃取大蒜精油，並利用氣相層析儀分析其DAS、DADS及DATS分別約佔總精油量的5、61及22%，並進一步以體外及體內試驗探討大蒜精油對人類黑色素細胞癌之抑制效果及分子機制。於探討大蒜精油抑癌機轉方面，大蒜精油可抑制人類黑色素癌A375細胞株之細胞存活率，並具有劑量與時間效應；流式細胞儀及西方墨點法之結果發現，大蒜精油會導致細胞週期停滯於G2/M期，並改變粒線體膜通透性（mitochondrial membrane permeability transition, MPT）及促進細胞內活性氧物質（reactive oxygen species, ROS）的生成，進一步啟動粒線體活化路徑，包括增加A375細胞Bax/Bcl-2的比值，促使caspase-9、caspase-3及poly(ADP-ribose)polymerase (PARP)明顯有裂解產物的表現，誘導A375細胞凋亡的發生。在細胞轉移實驗方面，大蒜精油具有降低基質金屬蛋白酶（matrix metalloproteinase, MMP）的活性，如：MMP-2與MMP-9，有效抑制A375細胞移動(migration)與侵襲(invasion)的能力。在細胞增生實驗部分，利用固著依賴性細胞菌落分析(anchorage-dependent colony formation assay)，大蒜精油可降低A375細胞株形成群落的能力，並經由MEK/MAPK [MAPK (mitogen-activated protein kinase)/ERK (extracellular signal regulated kinase) kinase]路徑抑制A375細胞增生。由以上結果證實，大蒜精油可藉由誘導A375細胞週期停滯G2/M期與細胞凋亡，並可抑制A375細胞株的轉移及增生。

本研究進一步以A375細胞株異種移植的模式，探討大蒜精油及DATS對黑色素細胞癌之抑制效果。將nu/nu裸鼠分為五組，分別為控制組 (葵花油0.1 mL)、大蒜精油低劑量組（58.8 mg/kg bw）與高劑量組(117.6 mg/kg bw)、DATS組(66.7 mg/kg bw)、正對照組（camptothecin i.v. 2 mg/kg bw ）。裸鼠分別管餵大蒜精油或DATS一週後，將A375細胞株以皮下注射方式接種於裸鼠中，實驗期間每兩天管餵一次，記錄其體重、攝食量與腫瘤體積的變化，連續九週後犧牲，並進行血液生化值的分析。結果顯示，大蒜精油高劑量組(117.6 mg/kg bw) 及DATS可有效抑制腫瘤體積與重量，對腫瘤體積之抑制率分別為62及84% (p＜0.05)，而對腫瘤重量之抑制率分別為45及83% (p＜0.05)；控制組與處理組之間的體重、肝功能指數(AST、ALT)及腎功能指數(BUN、Creatine) 皆沒有顯著差異，顯示大蒜精油及DATS並不會造成不良的影響。由細胞與動物實驗的結果推論，大蒜精油具有抑制黑色素細胞癌生長與轉移的作用之潛力。

關鍵字

大蒜 ； 黑色素細胞癌 ； 細胞凋亡 ； 細胞轉移 ； 異種移植

並列摘要

The incidence of melanoma increased more rapidly than any other cancer in recent years. Malignancies arising in the skin are broadly divided into two categories: melanoma and non-melanoma skin cancers. Melanoma is the most serious, highly invasive and metastatic skin tumor, and even resistant to chemotherapy. Numerous studies have shown that garlic (Allium sativum L.) oil and their major active compounds - diallyl sulfide (DAS), diallyl disulfide (DADS) and diallyl trisulfide (DATS) exhibit the anticancer activity against several cancer types, but their effects and related mechanisms of garlic oil on human melanoma cells were not clear yet. The objective of our study was to investigate the anticancer effect of garlic essential oil on human melanoma A375 cells in vitro and in vivo assays. Garlic essential oil is obtained by steam distillation which yield accounts for 0.2–0.5%. We have evaluated the profiles of allyl sulfides in garlic oil, including DAS (5%), DADS (61%), DATS (22%) using gas chromatography.
The results indicated that garlic oil inhibited cell viability in a dose dependent manner. Flow cytometry analysis and Western blot results showed garlic oil significantly induced G2/M phase arrest of A375 cells. We further demonstrated that garlic oil elicited apoptotic events such as mitochondrial membrane depolarization, increasing intracellular reactive oxygen species (ROS) generation, activation of caspase-9, caspase-3 and poly(ADP-ribose)polymerase (PARP). Additionally, garlic oil inhibited cell migration and invasion of A375 cells by in vitro anti-invasive assay. The activities and protein expressions of matrix metalloproteinase (MMP)-2 and -9 in A375 cells were inhibited by treatment with garlic oil. Moreover, we also showed that garlic oil suppressed anchorage-independent growth of A375 cells. The anti-proliferative potency might relate to inhibit phosphorylation of MEK/MAPK [MAPK (mitogen-activated protein kinase)/ERK (extracellular signal regulated kinase) kinase] pathway.
We also studied the anti-tumor activity of garlic oil and DATS in A375 tumor-bearing nude mice model. Garlic oil and DATS was administered for 1 week before inoculation of A375 cells and continued for 9 weeks. This results indicated that oral administration of garlic oil (117.6 mg/kg bw) and DATS (66.7 mg/kg bw) has potential to reduce tumor volume and weight as compared with negative control in xenografted nude mice; tumor volume was inhibited by 62 and 84% (p<0.05); tumor weight was inhibited by 45 and 83% (p<0.05), respectively. Furthermore, there was no significant difference in body weights among the various groups at the end of the study. The AST, ALT, BUN and creatine values showed no significant difference between treatment groups and control group. The results implied that garlic oil and DATS feeding to mice did not appear to induce any adverse effects.
In conclusion, garlic oil inhibited cell growth of human melanoma cells via induction of cell cycle arrest and apoptosis, and might provide the anti-metastatic and anti-proliferative potency. Therefore, these finding demonstrated that garlic oil and DATS exhibit anti-tumor effects in vivo model.
These results suggest that garlic oil provide the potential anticancer effect on anti-melanoma.

並列關鍵字

garlic ； melanoma ； apoptosis ； melanoma ； xenografted nude mice

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