羽扇豆醇增強DLD-1大腸癌細胞株對化療藥物5-氟尿嘧啶敏感性的機制探討

Abstract

大腸直腸癌的發生率高居全球的第三位，而其中氟尿嘧啶 (5-FU)是最廣泛被用來當作一線化療的用藥。然而該藥物常因癌細胞所產生的抗性及其高劑量毒性限制了它的藥物療效。因此找到一個能提升5-FU藥物作用的組合藥物是必需的。天然物中蘊藏著許多有潛力被發展成新穎藥物的分子，羽扇豆醇(lupeol)就是一個具有多元藥物活性的天然化合物，其中也包括了抗癌活性。即使過往已有多篇報導著墨於羽扇豆醇的抗癌研究，但科學家關注的是羽扇豆醇藉由抑制NFκB、 PI3K/Akt、 Wnt/β-catenin…等等的路徑來毒殺癌細胞，本論文研究則著重於其抑制UNG (uracil DNA glycosylase)的能力。我們的結果顯示，羽扇豆醇抑制UNG的現象是在後轉譯的層級而非在mRNA的層級，且其所造成的UNG不穩定性可能是藉由proteasome對其的降解所達成的。再者，此羽扇豆醇抑制UNG的現象也確實造成了細胞中UDG活性的下降。由於5-FU會讓癌細胞併入更多的尿嘧啶和氟尿嘧啶到細胞核內DNA中，進而造成DNA損傷。因此UNG移除這些錯誤鹼基的UDG能力就顯得格外重要。所以基於此羽扇豆醇抑制UDG的現象，我們決定以羽扇豆醇和5-FU共處理DLD-1這株大腸癌細胞株，看是否能因此提升5-FU的藥物作用。根據我們MTT試驗的結果以及CDI (Coefficient of Drug Interaction)值的計算可知，此兩藥物的組合治療確實會有增效結果產生(CDI < 1)。除此之外，羽扇豆醇與5-FU的組合也增加了更多的γ-H2AX，且在彗星試驗中觀察到更多具有彗星狀DNA損傷之細胞。這說明了此二藥物的協同作用可能是因為增加細胞中的DNA損傷所造成的。綜合以上發現，羽扇豆醇對UNG的抑制作用具有提升大腸癌細胞對5-FU化學治療藥物敏感性的潛力。

Colorectal cancer (CRC) is the third most common cancer in the world and fluorouracil (5-FU) has been wildly used as the first line therapy for it. However, the treatment effect is often hampered by the development of drug resistance or toxicity at high doses. Therefore a new candidate serving as a co-drug that can sensitize colorectal cancer cells to 5-FU is in need. Natural compounds represent a significant source for the development of novel cancer therapies. Lupeol is one of these natural compounds that exhibits versatile pharmacological activities including anti-cancer capability. Though it has been reported previously that the cytotoxic effects lupeol exerted on cancer cells may be through the inhibition of signaling pathways such as NFκB, PI3K/Akt, Wnt/β-catenin, we herein focused on its ability in downregulating UNG (uracil DNA glycosylase), an initiator of BER (base excision repair). Our results showed that lupeol suppressed UNG in protein level rather than in transcriptional level, and the stability of UNG was likely affected via proteasome degradation. Furthermore, the UDG activity was found to be decreased after lupeol treatment. In consideration of the phenomenon we have observed, we decided to co-treat DLD-1 colorectal cancer cell line with lupeol and 5-FU (5-fluorouracil). 5-FU is known to prime cells to incorporate more uracil or fluorouracil into the genome, triggering the DNA damage. Therefore, once accumulation of a large number of uracil or FU occurred, the importance of UNG level became prominent. Our data showed that co-treatment of lupeol and 5-FU on DLD-1 colorectal cancer cells resulted in a CDI < 1 (Coefficient of Drug Interaction), indicating a synergism. In addition, combination of lupeol and 5-FU increased γ-H2AX and comet tail was observed by single cell gel electrophoresis, indicating that the synergistic effect may be due to the increment of DNA damages in cells. These findings suggest that the UNG inhibition effect by lupeol has the potential in sensitization colorectal cancer to 5-FU.