高紹軒Shao-Hsuan Kao簡銘誼Ming-Yi Jian醫學研究所碩士班2026-10-292026-02-0120252025-01-16https://handle.ncl.edu.tw/11296/ay5rumhttps://scholars.csmu.edu.tw/handle/123456789/36728結直腸癌是全球第三大的癌症，根據世界衛生組織的統計，早期診斷並經過適當治療的患者，其五年存活率可高達90%以上。然而，由於早期症狀不明顯，許多患者在病情惡化時才被診斷，此時癌細胞可能已經轉移至其他器官，導致治療難度顯著提升，五年存活率驟降至僅5%至7%。結直腸癌的發病機制涉及基因突變、環境因素和生活習慣的綜合影響。儘管化療、放療和靶向治療等方法不斷發展，但傳統化療藥物的毒性和抗藥性問題，仍然是結直腸癌治療的重要挑戰。因此，開發新型輔助治療策略，尤其是與現有藥物的結合應用，成為結直腸癌的主要研究重點之一。順鉑是一種廣泛用於多種癌症治療的化療藥物，其主要機制是通過損傷細胞 DNA，誘導細胞凋亡。然而，順鉑治療會引發嚴重的副作用，如腎毒性和骨髓抑制等，並且長期使用容易導致癌細胞的抗藥性增強。積雪草酸是一種從積雪草（Centella asiatica）中提取的三萜類化合物，因其抗氧化、抗炎和抗腫瘤特性而受到廣泛關注。已有研究表明，積雪草酸能通過調控多條細胞信號通路，促進腫瘤細胞凋亡並減少順鉑引起的腎損傷。因此，本研究探討順鉑與積雪草酸的聯合作用是否能增強抗腫瘤效果，並減輕其毒性副作用。本研究以人類DLD-1 與 SW620 結直腸癌細胞株作為細胞模型，進行系統性評估順鉑與積雪草酸的合併使用的抗癌效用及其分子機制。實驗結果顯示，各別使用順鉑或積雪草酸均能降低結直腸癌細胞存活率，而合併處理可顯著提升抑制效果，包括增加細胞週期停滯與誘發細胞凋亡。細胞週期分析顯示，DLD-1 細胞在 sub-G1期的比例最高達39.2%，而SW620 細胞則可達8.6%，證實細胞週期停滯與凋亡的誘發。和單獨使用順博相比，Annexin V/PI 染色結果進一步證明合併處理能提高細胞凋亡率，其中DLD-1 細胞凋亡由17.03%增至35.3%，而SW620 細胞凋亡則由10.7%增至30.8%。西方墨點分析顯示，合併處理造成p53 蛋白表現上升，Cyclin D1 表現下降。此外，Cleaved Caspase-3、-8、-9 和 Cleaved PARP 表現在合併處理時有顯著增加，顯示內源性與外源性凋亡路徑均被活化。接著也發現促凋亡蛋白 Bax、Bim 表現上升，抗凋亡蛋白 Bcl-2 表現下降。活性氧（ROS）分析結果顯示，順鉑可顯著提升 ROS，而積雪草酸可能啟動其他抗氧化壓力機制，因此合併處理並未再進一步提高ROS。鐵凋亡標誌蛋白 GPX4 與 SLC7A11在合併處理時表現下降，推測可能產生脂質過氧化累積並誘導鐵凋亡。基因表現分析顯示，Bcl-2、SLC7A11、GPX4 mRNA 表現下降，並抑制細胞移動與侵襲能力，同時提升 E-cadherin 表現。本研究結果顯示，順鉑與積雪草酸的結合不僅能顯著促進結直腸癌細胞的凋亡，還能有效抑制其侵襲能力。這些發現為結直腸癌的輔助治療提供了潛在的新策略，並為未來的癌症治療開發提供了理論基礎。Colorectal cancer (CRC) is the third most common cancer in the world. Basing on the statistics from the World Health Organization, patients diagnosed early and treated appropriately can have a good five-year survival rate of > 90%. However, due to the lack of obvious early symptoms, many patients are usually diagnosed as late stage, at which point the cancer cells may have already metastasized to other organs, significantly increasing the difficulty of treatment, and causing the five-year survival rate to decline sharply to just 5% to 7%. The pathogenesis of colorectal cancer is complicated and affected by genetic mutations, environmental factors, and lifestyle habits. Despite the ongoing development of treatments for CRC such as chemotherapy and targeted therapy, the toxicity and resistance issues of traditional chemotherapy drugs remain significant challenges in CRC treatment. Therefore, the development of new adjuvant therapeutic strategies, especially in combination with chemotherapy drugs, has become one of the major research focuses in CRC. Cisplatin is a chemotherapy drug widely used in the treatment of various solid malignant tumors. The primary anticancer effect of cisplatin is attributed to induction of apoptosis by damaging cellular DNA and producing ROS. However, cisplatin often causes severe side effects, such as nephrotoxicity and bone marrow suppression, and long-term use can lead to enhanced drug resistance in cancer cells. Asiatic acid is a triterpenoid compound derived from Centella asiatica, which has garnered widespread attention due to its multiple biological benefits including antioxidant, anti-inflammatory, and anti-tumor activities. Previous studies have shown that asiatic acid promotes tumor cell apoptosis and reduce cisplatin-induced kidney damage by regulating diverse cellular signaling pathways. Therefore, this study explores whether the combination of cisplatin and asiatic acid enhance anti-tumor effects as compared to cisplatin alone. Human CRC cell line DLD-1 and SW620 were used as models. Our results showed that cisplatin or asiatic acid alone could reduce the survival rate of colorectal cancer cells, while the combined treatment significantly enhanced the inhibitory effects on CRC cells, including increasing cell cycle arrest and inducing apoptosis. Cell cycle analysis revealed that the proportion of DLD-1 and SW620 cells in the sub-G1 phase reached the maximum of 39.2% and 8.6%, respectively, indicating the induction of cell cycle arrest and apoptosis. Annexin V/PI staining further demonstrated that combined treatment increased the apoptosis rate compared to cisplatin alone, and the apoptosis rising from 17.03% to 35.3% (DLD-1) and from 10.7% to 30.8% (SW620). Western blot analysis showed that combined treatment resulted in upregulation of p53 protein and downregulation of Cyclin D1. Additionally, cleaved caspase-3, -8, -9, and cleaved Poly (ADP-ribose) polymerase (PARP) levels were significantly increased in response to the combined treatment, indicating activation of both intrinsic and extrinsic apoptosis pathways. Furthermore, the pro-apoptotic proteins Bax and Bim were found upregulated, while the anti-apoptotic protein Bcl-2 was downregulated. Reactive oxygen species (ROS) analysis using DCFh-DA showed that cisplatin significantly increased ROS, while the combined treatment did not further promoted ROS, which might attribute to the antioxidant activity of asiatic acid or the antioxidant signaling pathways activated by asiatic acid. We also explored the effect of Asiatic acid on ferroptosis, and the results revealed that GPX4 and SLC7A11 were decreased by the combined treatment, suggesting that asiatic acid may trigger the accumulation of lipid peroxidation and induction of ferroptosis. In parallel to protein level evaluation, gene expression analysis revealed that the combination treatment downregulated Bcl-2, SLC7A11, and GPX4 mRNA, inhibited cell migration and invasion, and upregulated E-cadherin expression. The results of this study indicate that the combination of cisplatin and asiatic acid not only significantly promotes apoptosis in CRC cells, but also effectively inhibits their invasiveness. Taken together, these findings provide evidences supporting that Asiatic acid has potent anticancer activity against CRC cells, and the combination of Asiatic acid with cisplatin promotes the cisplatin’s cytotoxicity. It suggests that Asiatic acid is a potential adjuvant therapy for CRC treatment.結直腸癌積雪草酸順鉑細胞凋亡鐵凋亡Colorectal CancerAsiatic AcidCisplatinApoptosisFerroptosisthesis