探討薑黃素與UVB對於人類黑色素瘤細胞株A375之影響

The Effects of Combined Curcumin and UVB on A375 Cell Lines

黃于綺(Yu-Ci Huang)

指導教授 ： 張基隆

高雄醫學大學/醫學院/醫學系生物化學科碩士班/碩士(2015年)

摘要

光照治療 (Phototherapy)是指以紫外線為光源來做治療，紫外線波長在約200~400nm範圍。其中又分UVA、UVB和UVC等不同波段。光波長不同會影響照射皮膚深度的差異。其中UVB主要影響表皮層的角質細胞 (Keratinocyte)，因此在臨床上常被應用於皮膚病的治療。薑黃素(Curcumin)主要來自於薑黃(Turmeric)根莖部，為提供咖哩食材的主要成份及顏色色素，同時也是常見的食品添加物及天然色素。近幾年更發現薑黃素對於抗氧化有顯著的功效，此外，亦有文獻指出薑黃素作用於癌細胞會誘導細胞凋亡，並顯示薑黃素具抗癌作用。文獻也指出薑黃素可作為一種光敏劑用以加強UVB對於人類皮膚角質細胞株HaCaT的作用。薑黃素與UVB對於人類皮膚癌細胞的作用目前仍沒有明確的分析研究。因此本研究重點為探討薑黃素結合UVB對人類黑色素瘤細胞株A375之影響。研究結果發現人類黑色素瘤細胞株A375分別單獨給予薑黃素及UVB照射時，隨著劑量的增加其細胞死亡率也明顯增加。而在同時給予薑黃素及UVB照射處理，其人類黑色素瘤細胞株A375之細胞死亡率，相較於單獨給予薑黃素及UVB照射明顯增加。同時給予薑黃素及UVB照射處理下，發現人類黑色素瘤細胞株A375之細胞型態明顯改變，且會導致人類皮膚癌細胞DNA片段化，並增加Sub-G1的細胞數目，及ATP減少等情形發生。更進一步研究發現，同時給予薑黃素及UVB照射處理下，會使促細胞凋亡蛋白Bax表現增加，抗細胞凋亡蛋白Bcl-2表現受到抑制，並活化Pro-caspase 3及Pro-caspase 9，進而導致人類皮膚癌細胞走向細胞凋亡。我們進一步釐清薑黃素結合UVB對於人類黑色素瘤細胞株A375能量代謝之影響，以探討其走向細胞凋亡之原因。發現薑黃素結合UVB作用於人類黑色素瘤細胞株A375後，會使促細胞內檸檬酸(Citrate)有堆積現象，且會促進抑制醣解相關蛋白TIGAR (TP53-inducible glycolysis and apoptosis regulator)及PDK (Pyruvate dehydrogenase kinase)表現增加，促醣解相關蛋白Hexokinase及PDH-E1Pyruvate dehydrogenase)受到抑制，導致ATP無法生成；同時也發現給予薑黃素及UVB照射處理下，會促使葡萄糖轉運蛋白Glut1 (Glucose transporter 1) 及G6PD (Glucose-6-phosphate dehydrogenase)表現增加，以增加葡萄糖之攝取，並促使ATP大量耗損，進而促使人類黑色素瘤細胞走向細胞凋亡。綜合上述結果得知，薑黃素可增加UVB對於黑色素瘤細胞細胞之毒殺能力，並導致細胞死亡，證實薑黃素結合UVB照射具有治療黑色素瘤細胞之潛力。

關鍵字

薑黃素 ； UVB ； 人類黑色素瘤細胞株

並列摘要

Phototherapy is to expose skin in ultraviolet light (UV) on a regular basis and under medical supervision. The UV, ranged from 10 to 400 nano-meter, are divided into 3 groups of UVA, UVB, and UVC; and UVB radiation is the most common form of phototherapy and worldwide used to treat skin diseases especially for keratinocyte. UVB can penetrate the skin and slow the growth of affected skin cells and that has been proved in many other skin conditions. Curcumin, a substance in turmeric, is a polyphenol and commonly used as coloring agent and food additive. Extensive research has indicated this kind of polyphenol can be used for prevention and treatment of cancer. Previous studies indicated that curcumin might be a promising photosensitizer to induce apoptosis on human keratinocyte cell line HaCaT and there was synergistic effect of UVB and curcumin on induction of apoptosis in HaCaT cells. Up to now, the effects of combined curcumin and UVB on skin cancer cell, such as malignant melanoma, is still unknown.
The present study was designed to investigate the effects of the combination of curcumin and UVB on human malignant melanoma cell line A375 and to explore the underlying mechanism. Our data showed that, after 24 h treatment with UVB irradiation or curcumin, the cell viability of A375 cells was decreased. Meanwhile, curcumin could enhance the cytotoxicity of UVB treatment. After co-treatment with75 mJ/cm2 UVB and 10 μM curcumin for 24h, cell morphology was changed and an oligonucleosomal DNA ladder was observed. Besides, the distribution of Sub-G1 in cell cycle, expression of apoptotic protein, Bax, and activation of pro-caspase-3 and pro-caspase-9 were increased; while expression of anti-apoptotic protein, Bcl-2 was decreased. These results indicated this combination stimulated A375 cell undergoing apoptosis. Furthermore, we found the combination would make citrate accumulation and ATP levels decrease which was owing to the disturbance of glycolysis by increasing in expression of Glut 1(Glucose transporter 1), G6PD (Glucose-6-phosphate dehydrogenase), T1GAR (TP53-inducible glycolysis and apoptosis regulator) and PDK (Pyruvate dehydrogenase kinase) and reuction in expressions of Hexokinase and PDH-E1Pyruvate dehydrogenase). Accordingly, we proposed that the induction of apoptosis by the combination is due to the decreased ATP production which resulted from the disturbance of glucose metabolism.
Results of this study suggest that the combined treatment of curcumin and UVB has the potential for skin cancer therapy.

並列關鍵字

curcumin ； UVB ； A375

參考文獻

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