| 摘要: | 大腸直腸癌（CRC）是全球最常見的惡性腫瘤之一，也是癌症相關死亡的主要原因。雖然現有治療有所進步，但抗藥性、腫瘤復發及副作用仍限制其療效，因此尋求新穎的替代療法勢在必行。傳統中醫藥（TCM）因其抗癌潛力受到廣泛關注，其中檳榔 (Areca catechu) 萃取物（ACE）已被發現對多種癌症細胞具有細胞毒性作用。本研究透過體外、體內實驗及轉錄體分析，探討 ACE 及其主要活性成分檳榔鹼（Arecoline）對 CRC 的抗癌機制。體外實驗顯示，ACE 以濃度依賴性方式顯著降低 CRC 細胞存活率。Annexin V 染色及流式細胞儀分析證實其可誘導細胞凋亡，並增加晚期凋亡細胞比例。Western blot 分析顯示，ACE 可下調抗凋亡蛋白PIK3CA、PIK3R1、p-AKT1 及 BCL2。此外，ACE 處理可顯著提高細胞內活性氧（ROS）水準，而預先使用抗氧化劑 N-乙酰半胱氨酸（NAC）可有效抑制 ROS 累積，顯示氧化壓力在 ACE 誘導的細胞凋亡過程中發揮關鍵作用。次世代定序（NGS）分析鑑定出 2,533 個上調基因及 2,130 個下調基因，顯示 ACE 透過調控 Wnt、PI3K/AKT 及 MAPK 訊息傳遞途徑影響腫瘤進展。基因本體論（GO）分析發現，上調基因參與細胞凋亡、DNA 損傷及自噬等生物過程，而細胞增殖及細胞週期調控相關基因則顯著下調。
在裸鼠異種移植模型中，ACE 處理可輕微抑制腫瘤生長，但未達統計顯著性。重要的是，組織病理學分析顯示肝臟與腎臟無明顯毒性影響，表明 ACE 具有良好的安全性。 此外，本研究鑑定檳榔鹼為 ACE 的主要活性成分，並發現其對 CRC 細胞展現強效細胞毒性。檳榔鹼可誘導細胞凋亡、導致 G2/M 期細胞週期停滯，並顯著抑制細胞遷移。轉錄體分析顯示，檳榔鹼可下調 VEGF 訊息傳遞相關基因，推測其可能參與腫瘤血管新生的調控。功能實驗進一步證實 VEGF-C 可部分逆轉檳榔鹼誘導的細胞凋亡並恢復細胞遷移，確認 VEGF 訊息傳遞途徑在檳榔鹼抗癌作用中的關鍵角色。
綜合而言，本研究揭示 ACE 及檳榔鹼的抗癌分子機制。ACE 透過誘導氧化壓力、啟動細胞凋亡訊號並抑制癌基因相關訊息傳遞發揮抗癌作用，而檳榔鹼則透過調控 VEGF 訊息傳遞抑制腫瘤進展。這些結果支持 ACE 和檳榔鹼作為潛在的 CRC 治療候選藥物，未來應進一步研究以優化其臨床應用。;Colorectal cancer (CRC) is among the highly prevalent identified oncologic diseases and remains a major factor of cancer- associated death rate on a global scale. Despite progress in routine therapies, complications such as drug resistance, tumor recurrence, and serious side highlight the need for alternative therapeutic strategies. Traditional Chinese Medicine (TCM) has garnered recognition for its potential anticancer properties, and Areca catechu extract (ACE) has demonstrated cytotoxic activity against various cancer types. This investigation intended to investigate the anticancer mechanisms of ACE and its major bioactive alkaloid, arecoline, in CRC through in vitro, in vivo, and transcriptomic analyses. ACE administration significantly suppressed CRC cell viability proportionally to the dosage. Flow cytometry with Annexin V staining confirmed apoptosis induction, while western blot analysis revealed ACE-mediated downregulation of proteins involved in cell survival, including PIK3CA, PIK3R1, p-AKT1, and BCL2. Additionally, ACE elevated intracellular reactive oxygen species (ROS) levels, and pre-administration with N-acetylcysteine (NAC) reduced ROS accumulation, indicating oxidative stress involvement in ACE-induced apoptosis. Next-generation sequencing (NGS) identified 2,533 upregulated and 2,130 downregulated DEGs, implicating ACE in major oncogenic pathways, for instance Wnt, PI3K/AKT, and MAPK signaling. Gene ontology (GO) analysis highlighted apoptosis, DNA damage, and autophagy among upregulated DEGs, while pathways linked to cell proliferation and the cell cycle were suppressed. In vivo, ACE administration slightly suppressed tumor size in nude mice xenograft model, though the effect was not significant. Histopathological analysis confirmed no ACE-induced toxicity. Additionally, arecoline was identified as an active component responsible for ACE’s anticancer effects, inducing apoptosis, G2/M phase arrest, and restraining cell migration. Transcriptomic and functional assays confirmed arecoline’s involvement in VEGF signaling modulation. These findings suggest ACE and arecoline as potential therapeutic agents for CRC by modulating oxidative stress, apoptosis, and tumor progression pathways, warranting further investigation for clinical applications. |
