皮膚美白藥物在人類惡性黑色素瘤細胞的藥物學上造成的影嚮

Pharmacological Effect of Skin Whitening Agents on Human Malignant Melanoma Cells

鄭森隆(Sun Long Cheng)

指導教授 ： 蔡嘉哲

中山醫學大學/醫學院/醫學研究所/博士(2007年)

https://doi.org/10.6834/CSMU.2007.00074

摘要

長久以來，化妝品調製技術中一向用麴酸kojic acid來美白或漂白皮膚。從毒物學的角度來看，沒有任何報導指出口服麴酸kojic acid會造成急性或亞慢性中毒，但是注射麴酸kojic acid的確有可能引起抽搐被提出。此外，有部份的報告評估麴酸kojic acid致癌的可能性以及使用麴酸kojic acid美白皮膚造成基因毒性變異的風險。
然而，高劑量的麴酸kojic acid會帶來副作用。最近，麴酸kojic acid已被証實有致癌性。在實驗中發現白鼠身上的「肝癌細胞形成之因子」會因此而增加。不過，使用極小劑量的麴酸kojic acid來美白皮膚並不會對使用者造成任何顯著性的基因變異。
雖然目前已有許多研究專注於麴酸kojic acid對人體或其他生物體造成的致癌性及基因毒性，麴酸kojic acid對正常及異常疾病的基因表現與調控機制卻還很少被研究。
熊果素arbutin是一種天然提煉的對笨二酚衍生物。目前熊果素arbutin是一種常見的美容保養品原料。臨床上，熊果素arbutin用於治療皮膚色素沉澱及抑制黑色素生成的效果顯著。但近年來的調查結果讓人們開始擔心熊果素arbutin的安全性及副作用。雖然目前已有報告研究說明熊果素arbutin抑制黑色素生成的成效與機制，但是我們對於在人類基因體層級下，熊果素arbutin對於惡性皮膚黑色素瘤產生的生物效能機制並不清楚且少有研究。
因為使用熊果素arbutin治療惡性皮膚黑色素瘤的基因表現從未被報導，本文使用基因微陣列DNA microarray顯色分析系統來研究熊果素arbutin對於惡性皮膚黑色素瘤的抗癌效果非常的富於挑戰。
對於麴酸kojic acid和熊果素arbutin作用在A375黑色素瘤細胞中,使用高產能基因微陣列和生物資訊庫分析不同表現基因的反應。
不同基因的改變，使用RT-qPCR反轉錄cDNA來確認分析，證實麴酸kojic acid和熊果素arbutin表現量以符合DNA的微陣列的分析，經證實的基因在生物標記得到正確資料。
使用基因微陣列顯色分析系統分析麴酸kojic acid治療黑色素瘤A375細胞之後，我們可以發現有433種不同顯現的基因（約有361種基因可列出其功能性，包含有136種上調表達基因up-regulated genes和225種下調表達基因down-regulated genes。）其中，在利用麴酸kojic acid治療黑色素瘤A375細胞之後，有7種下調表達基因（APOBEC1，ARHGEF16，CD22，FGFR3，GALNT1，UNC5C，ZNF146）適合篩選作為腫瘤抑制基因。這些基因可能使惡性皮膚黑色素瘤的活動性消失，並且成為未來診斷及治療應用的實用標記。我們的研究同時列出使用麴酸kojic acid來治療免疫系統異常、骨骼發育、骨骼保養時可能出現的副作用與缺點。不過，我們需要進一步研究麴酸kojic acid對人體皮膚細服的生物與分子機制。同時，我們也必需驗視麴酸kojic acid用在臨床治療時表現出的其他生物學特性。
使用基因微陣列顯色分析系統分析熊果素arbutin治療黑色素瘤A375細胞之後，我們可以發現有357種不同顯現的基因（約有324種基因可列出其功能性，包含有88種上調表達基因up-regulated genes和236種下調表達基因down-regulated genes。）其中，在利用熊果素arbutin治療黑色素瘤A375細胞之後，有4種下調表達基因（AKT1，CLECSF7，FGFR3和LRP6）被篩選認為可能是抑癌基因。這些基因可能使惡性皮膚黑色素瘤的活動性消失，並且成為未來診斷及治療應用的實用標記。
熊果素arbutin與麴酸Kojic acid對黑色素瘤A375細胞造成的細胞基因毒理學上變異性相似。這也許表示兩者對惡性腫瘤發生的調控效果相似。
然而，我們應該進一步研究熊果素arbutin對人體皮膚細胞的生物與分子機制。同時，我們也必需檢驗arbutin熊果素用在臨床治療時表現出的其他生物學特性。

關鍵字

麴酸 ； 熊果素 ； 惡性黑色素瘤細胞 ； 微陣列

並列摘要

Kojic acid (5-hydroxy-2-hydroxymethyl-1,4-pyrone) is as a skin lightening or bleaching agent for a long time in cosmetic preparations. About the toxicological aspect, the acute or subchronic toxicity resulting from an oral dose of kojic acid has never been reported, but convulsions may occur if kojic acid is injected. Moreover, some reports showed the evaluation of the tumorigenic potential of kojic acid and study the genotoxic risk for humans using kojic acid as a skin-lightening agent.
On the other hand, kojic acid at high dose has some side effects; Recently, kojic acid is found to be a tumor promoter and can also enhance the hepatocarcinogenesis in rat as well as in mice. Moreover, the topical use of kojic acid as a skin lightening agent results in minimal exposure that poses no or negligible risk of genotoxicity or toxicity to the consumer.
Although kojic acid has been widely studied in the tumorigenic potential, genotoxic risk for humans and other applications, the mechanism of kojic acid on the gene expression and regulation in normal and/or diseased cells has rarely been studied.
Arbutin, a natural compound of beta-D-glucopyranoside of hydroquinone, is widely used as an ingredient in skin care products. It is effective in the treatment of various cutaneous hyperpigmentations and has the inhibitory effects on the melanogenesis in melanoma cells.
Recent findings have raised serious questions of concern regarding both the safety and side-effect of arbutin. Although some inhibitory effects of arbutin on melanogenesis in melanoma cells and its mechanism have been elucidated, the intensive study of its biological effect on human genomics level in the regulation of malignant melanogenesis through the functional effect on carcinogenesis is not clear and rarely reported.
Since the study of gene expression profiling in human melanoma cells treated with arbutin has never been reported, it would be very challenging to use the DNA microarray in studying the biological effect of arbutin on malignant melanoma cells through its action on anti-cancer property.
We used the high throughput analysis of DNA microarray and bioinformatic tools for a global analysis of differentially expressed genes responding to kojic acid and arbutin in A375 malignant melanoma cells.
The changes of differentially expressed genes, which were validated by RT-qPCR analysis, gave the confirmatory quantitative results under stringent condition and revealed that the quantity or expression level of kojic acid and arbutin-responsive genes agreed with the DNA microarray data. It indicated that the validated genes in the robust biomarkers lists obtained precise data.
The DNA microarray analysis of kojic acid-treated A375 cells revealed that the total number of 433 differentially expressed probes was matched with approximately 361 listed genes, containing 136 up-regulated genes and 225 down-regulated genes. Seven down-regulated genes (APOBEC1, ARHGEF16, CD22, FGFR3, GALNT1, UNC5C and ZNF146) served as candidate tumor suppressor genes in A375 cells after kojic aicd treatment. They may deactivate the regulation of malignant tumorigenesis in human malignant melanoma cells and may become useful markers for further diagnostic and therapeutic applications. Our findings also showed the probably side effects of disadvantages of kojic acid on the dysfunction of immune system and bone development & maintenance.However, we will conduct further studies on the effects of kojic acid on the biological and molecular mechanisms of human skin cells, and also examine other biological characterizations of kojic acid for therapeutic applications.
The DNA microarray analysis of arbutin-treated A375 cells revealed that the total number of 357 differentially expressed probes was matched with approximately 324 listed genes, containing 88 up-regulated genes and 236 down-regulated genes.
Four down-regulated genes (AKT1, CLECSF7, FGFR3 and LRP6) served as candidate tumor suppressor genes in A375 cells after arbutin treatment. They may deactivate the regulation of malignant tumorigenesis in human malignant melanoma cells and may become useful markers for further diagnostic and therapeutic applications.
The genotoxic effect of arbutin on gene expression profiling of A375 cells was similar to kojic acid effect, which may indicate the similar regulation of malignant tumorigenesis.
However, we will conduct further studies on the effects of arbutin on the biological and molecular mechanisms of human skin cells, and also examine other biological characterizations of arbutin for therapeutic applications.

並列關鍵字

kojic acid ； arbutin ； Malignant Melanoma Cells ； microarray

參考文獻

2. Aggarwal B. B., Bhardwaj A., Aggarwal R. S., Seeram N. P., Shishodia S. and Takada Y., Role of resveratrol in prevention and therapy of cancer: preclinical and clinical studies. Anticancer Res. 24: 2783-2840, 2004.

1.Ackerman, S. L., Knowles, B. B., 1998. Cloning and mapping of the UNC5C gene to human chromosome 4q21-q23. Genomics 52, 205-208.

3.Ahn, K. S., Moon, K. Y., Lee, J., Kim, Y. S., 2003. Down regulation of NF-kB activation in human keratinocytes by melanogenic inhibitors. Journal of Dermatological Science 31, 193-201.

4.Arakawa, H., 2004. Netrin-1 and its receptors in tumorigenesis. Nature Reviews 4, 978-987.

5 Arce I., Roda-Navarro P., Montoya M. C., Hernanz-Falcon P., Puig-Kroger A. and Fernandez-Ruiz E., Molecular and genomic characterization of human DLEC, a novel member of the C-type lectin receptor gene family preferentially expressed on monocyte-derived dendritic cells. Eur. J. Immunol. 31: 2733-2740, 2001.