綠原酸合併順鉑在抑制大腸癌細胞增殖與轉移之分子機轉研究

大腸直腸癌(Colorectal Cancer, CRC)為全球發生率第三高的癌症。近年 來，天 然多酚類化合物因具備抗氧化、抗癌及調控細胞訊息傳遞等生物活 性而備受關注。其中，綠原酸(Chlorogenic Acid, CGA)為廣泛存在於咖啡與 蔬果中的天然多酚，具潛在抗癌效果。本研究旨在探討 CGA 與常用化療藥 物順鉑(Cisplatin, CDDP)聯合處理對大腸癌細胞株 DLD-1 的影響。透過 MTT 與 SRB 細胞存活率分析發現，CDDP 與 CGA 單獨皆具抑制細胞生 長之能力，且呈現劑量依賴性;合併處理則展現明顯提升的細胞毒性效應。 流式細胞儀分析細胞週期分布的結果顯示，合併處理組中 Sub-G1 週期比例 顯著上升，代表凋亡細胞增加。西方墨點法進一步驗證，相較於單獨處理， 合併處理增加凋亡促進蛋白 Bim、活化型 Caspase-3 與 cleaved PARP 的 表現，同時降低抗凋亡蛋白 Bcl-2 與 XIAP 的表現，顯示合併處理可提升 內源性凋亡路徑的活化以及調降凋亡抑制因子。此外，合併處理顯著提升細 胞內活性氧(ROS)含量，因此進一步探討是否誘發鐵凋亡。然而，鐵凋亡抑 制劑前處理無顯著回復合併處理所降低的細胞存活率，且粒線體運鐵蛋白 TfR-1 表現無明顯改變，而 GPX4 與 xCT 表現卻呈現上升的現象，顯示合 併處理可能透過增加其他 ROS 介導的細胞死亡機制，而非誘發鐵凋亡，詳 細機轉尚待釐清。另一方面，使用廣效 caspase 抑制劑 Z-VAD-fmk 可回復部分細胞存活率，推測合併處理主要為誘發細胞凋亡。綜合實驗結果顯示， CGA 與 CDDP 合併處理可誘發氧化壓力與活化凋亡訊號，有效抑制大腸癌 細胞存活，顯示 CGA 具有作為提升大腸直腸癌化療功效之潛力。

Colorectal cancer (CRC) is the third most common malignancy worldwide. Natural polyphenolic compounds have garnered increasing attention in recent years due to their antioxidant, anticancer, and cell signaling regulatory properties. Among them, chlorogenic acid (CGA), a naturally occurring polyphenol abundant in coffee and various fruits and vegetables, has demonstrated potential anticancer effects. This study aimed to investigate the effects of CGA in combination with the chemotherapeutic agent cisplatin (CDDP) on the colorectal cancer cell line DLD-1. MTT and SRB assays revealed that CDDP and CGA individually suppressed cell viability in a dose-dependent manner, while their combined treatment exhibited significantly enhanced cytotoxicity. Flow cytometric analysis showed a marked increase in the sub-G1 population in the combination group, indicating an elevation in apoptotic cells. Western blot analysis further demonstrated that the combination treatment upregulated pro-apoptotic proteins such as Bim, activated caspase-3, and cleaved PARP, while downregulating anti- apoptotic proteins Bcl-2 and XIAP, suggesting activation of the intrinsic apoptotic pathway. In addition, the combined treatment significantly increased intracellular reactive oxygen species (ROS) levels. Therefore, the involvement of ROS- mediated ferroptosis was then explored. However, pretreatment with the ferroptosis inhibitor failed to restore cell viability, and no significant changes in transferrin receptor 1 (TfR-1) expression were observed. Conversely, expression levels of GPX4 and xCT were elevated, implying that other ROS-mediated cell death rather than ferroptosis was provoked. The underlying mechanisms remain to be elucidated. Furthermore, partial recovery of cell viability was achieved by pretreatment with the pan-caspase inhibitor Z-VAD-fmk, indicating that apoptosis is the primary mode of cell death induced by the combination therapy. These findings suggest that CGA combined with CDDP induces oxidative stress and activates apoptotic signaling pathways, thereby effectively inhibiting the survival of colorectal cancer cells. CGA may thus serve as a potential adjuvant to enhance the efficacy of chemotherapy in CRC treatment.