探討抗壞血酸(維他命C)與順鉑聯合治療對人類食道鱗狀細胞癌之抗癌活性

Abstract

食道癌(Esophageal cancer, EC)目前在全球最致命的癌症之一，主要因為食道癌的預後和生存率都很差。目前食道癌治療方法包括外科手術，放射線和化學療法的複合性治療。順鉑(Cisplatin, CDDP)是用於食道癌治療常見的化學藥物之一，CDDP的細胞毒性是通過形成活性氧化物(Reactive oxygen species, ROS)與形成順鉑-DNA加合物導致無法修復的DNA損傷。然而，高副作用和抗藥性的產生成為食道癌治療的障礙。近年來研究指出藥理濃度的抗壞血酸(Ascorbic acid, AA)可以殺死癌症細胞而不會傷害正常的細胞。在本研究中，將找出一種可能增強食道癌對化療敏感性的新藥物組合策略。我們的目標是1)評估CDDP和AA聯合治療鱗狀食道細胞癌(ESCC)的效果； 2)了解CDDP和AA聯合治療ESCC的作用機制。首先，使用MTT(3-(4,5-二甲基噻唑-2-基) -2,5-二苯基四唑-溴化物)評估CDDP和AA組合對細胞存活和菌落形成的協同作用。數據顯示，高劑量AA治療可以殺死ESCC，並且CDDP和AA協同殺死ESCC和降低ESCC的菌落形成能力。接下來，我們觀察到CDDP-AA組合會造成鱗狀食道癌細胞週期sub G1積累並且導致更多的細胞凋亡。藉由流式細胞儀分析結果，可以證實CDDP-AA組合誘導鱗狀食道癌細胞中的氧化壓力和粒線體功能下降，例如ROS上升和粒線體膜電位的下降。然而，經過活性氧化物清除劑N-acetylcysteine (NAC)前處理，結果證實CDDP-AA組合所誘導鱗狀食道癌細胞的變化都是由氧化壓力造成。在基因表現的部分，CDDP-AA聯合治療調控在細胞凋亡途徑上與粒線體相關的Bcl2蛋白質家族，降低抗凋亡基因並增加凋亡基因的表現，通過了解CDDP-AA聯合治療對鱗狀食道癌細胞的作用和機制，CDDP-AA聯合治療將成為提高清除鱗狀食道癌的治療效果有潛力的候選者。

Esophageal cancer (EC) is one of the most lethal cancers worldwide with poor prognosis and survival rate. The treatment of EC patients includes surgery, followed by radiotherapy and chemotherapy. Cisplatin (CDDP) is one of the common chemical drugs used in the treatment of EC, and the cytotoxicity of CDDP is through the formation of reactive oxygen species (ROS) and the formation of cisplatin-DNA adducts leading to irreparable DNA damage. However, serious side effects and drug resistance stand as a barrier to the therapeutic efficacy of EC. Many studies had pointed out that pharmacological ascorbic acid (AA) can kill cancer cells without injured to normal cells. In this study, we wanted to identify a novel drug combination strategy that could increase the sensitivity of EC to chemotherapy. I aimed to 1) evaluate the effects of CDDP and AA treatment in esophageal squamous cell carcinoma (ESCC); 2) understand the mechanism of CDDP and AA combine treatment in ESCC. First, the synergistic effect of the CDDP and AA combination was evaluated using MTT assay and colony formation assay. The data showed that high-dose AA treatment could kill ESCC cells, and that CDDP and AA synergistically killed ESCC cells and reduced the colony-forming ability of ESCC cells. Next, we observed that the CDDP-AA combination caused ESCC sub G1 arrest and led to more apoptosis. By flow cytometry analysis of the results, it was confirmed that the CDDP-AA combination induced oxidative stress and decreased mitochondrial function in ESCC cells, such as increased ROS level and decreased mitochondrial membrane potential. However, the results of after pretreatment with the reactive oxide scavenger N-acetylcysteine (NAC) in cells validated that all CDDP-AA combination induced changes in ESCC cells were triggered by oxidative stress. Finally, CDDP-AA combination therapy regulated the mitochondria-related Bcl2 protein family in the apoptosis pathway, reduced anti-apoptotic genes and increased the expression of apoptotic genes. By understanding the role and mechanism of ESCC cells, CDDP-AA combination therapy will be a promising candidate to improve therapy efficacy for eliminating esophageal cancer.