以基因體學與蛋白質體學探討大蒜精油及二丙烯基三硫化物抑制人類惡性黑色素瘤細胞之可能分子機制

Abstract

惡性黑色素瘤（malignant melanoma），由黑色素細胞癌化而成，為一種死亡率相當高的皮膚癌。由於黑色素細胞瘤的每個細胞都有成為癌症幹細胞的潛力，具有高侵襲及轉移能力，且難以手術或其他癌症療法治療，研究發現近年來西方國家及台灣的發生率都有逐年上升的情形，因此需要找尋預防方式或輔助性療法以改善黑色素瘤的發生或進展。大蒜精油具有許多種活性成分，對多種癌細胞株具有抑制活性，如：丙烯基硫化物（allyl sulfides），在丙烯基硫化物中更以二丙烯基三硫化物（diallyl trisulfide, DATS）效果最佳。然而，目前大蒜精油與丙烯基硫化物對人類皮膚癌細胞影響的相關研究卻很少見，先前本實驗室研究以不同劑量之大蒜精油和其純物質DATS餵食具有A375黑色素細胞株異種移植之裸鼠，發現高劑量大蒜精油（117.6 mg/kg bw）與DATS（66.7 mg/kg bw）能使裸鼠體內之異種移植腫瘤體積下降，因此本研究進一步利用DNA微陣列晶片及二維膠體電泳等體學研究方法，配合系統生物學軟體，了解大蒜精油及DATS抑制A375異種移植黑色素瘤之可能機制。結果指出，因大蒜精油的分析結果較不顯著，乃以純物質DATS作為主要探討對象。基因體學與蛋白質體學分析結果顯示，DATS能夠降低HMGB1蛋白、細胞激素IL-6、IL-1β及趨化素CCL2、CCL20等免疫反應相關基因及蛋白之表現量，減少腫瘤組織內發炎微環境；抑制糖解作用酵素ALDOC、TPI1、PGAM1、PKM2表現，減少癌細胞利用糖解作用獲得能量；降低泛素化系統相關酵素蛋白表現，降低抑癌相關蛋白質的降解情形；抑制hsp27及gelsolin抑制細胞凋亡之蛋白表現，促進腫瘤細胞凋亡；減少癌細胞轉移相關蛋白vimentin、annexin A4、gelsolin、Arp2/3、profilin 2，以及ECM重塑相關基因collagenI、collagen III、MMP-9表現。綜合以上所述，DATS可能透過抑制發炎相關之免疫反應、蛋白質泛素化系統、糖解作用路徑，並促進細胞凋亡，進一步抑制A375異種移植腫瘤的生長或轉移。以上結果推測大蒜精油及DATS具預防黑色素瘤之潛力，未來可作為預防或是輔助黑色素瘤治療方法。

Malignant melanoma is cancerous from melanocyte which is the most deadly kind of skin cancer. Since each melanoma cell has potential to become stem cell, it with highly invasive and metastasis characteristic, and it’s hard to cure by surgery or other cancer therapy. Some studies have found that the incidence of melanoma in western countries or Taiwan has increased year by year; therefore it is important to find the ways of prevention or adjuvant therapy to improve melanoma incidence or progression. Diallyl trisulfide (DATS), the most active component of garlic essential oil (GO), has been reported to provide antitumor activity in several cancer types. However, the actually related mechanism of GO and DATS on skin cancer is still unclear. Towards this, our lab has done the xenograft model of human melanoma in nude mice, which was established by injecting A375 cells. Tumor-induced mice were orally administered with GO (117.6 mg/kg bw) and DATS (66.7 mg/kg bw) respectively, and observed for the anti-skin tumor effects for nine weeks. The results revealed that tumor size was significantly reduced by GO and DATS treatment. The objective of this study is using genomics and proteomics technologies to investigate the molecular mechanisms of GO and DATS against human melanoma A375 cells in tumor xenograft models. Gene expression data from microarray and the differentially expressed proteins identified through 2D gel electrophoresis were integrated to elucidate the possible mechanisms of anti-tumor property. Since garlic oil analysis results are less significant, we turn to focus on DATS. Possible mechanisms of DATS against A375 induced xenograft tumor at genomics and proteomics levels revealed that DATS can reduce HMGB1 protein, cytokine IL-6, IL-1β, chemokines CCL2, CCL20 and other immune response genes and proteins expression, reducing inflammation within the tumor tissue microenvironment; DATS inhibiting glycolysis enzymes ALDOC, TPI1, PGAM1, PKM2 performance, reduce cancer cells use glycolysis to obtain energy; DATS decrease ubiquitin system-dependent enzyme protein expression, reducing tumor suppressor protein degradation-related situations; DATS inhibiting hsp27 and gelsolin apoptosis-inhibited proteins expression, and promote tumor cell death; DATS reduce metastasis-associated protein vimentin, annexin A4, gelsolin, Arp2/3, profilin 2, and ECM remodeling genes collagenI, collagen III, MMP-9 expression. In conclusion, DATS is related to inflammation by inhibiting the immune response, ubiquitination system, glycolysis path and promote apoptosis, further suppressed A375 xenograft tumor growth or metastasis. These results suggest that DATS has the potential to prevent melanoma, and can be used as secondary prevention or treatment of melanoma in the future. These results give evidences for the anti-skin tumor activity of GO’s active compound, DATS.