探討檸檬醛合併順鉑處理對口腔癌細胞遷移抑制及化療增敏之效用及其分子機制

口腔癌具有高度的侵襲性，由於早期診斷不易且現有治療策略成效不彰，因此患者預後不佳、死亡率高。順鉑 (cisplatin)為臨床常用口腔癌化療藥物，但其副作用與癌細胞的抗藥性限制了其療效。近年研究指出，檸檬醛 (citral)在多項癌症中皆有抗癌能力與腫瘤選擇性，因此受到廣泛關注。然而在口腔癌領域的研究迄今仍相當有限。因此本研究旨在探討天然化合物檸檬醛是否能抑制口腔癌細胞遷移能力，並合併順鉑使用，評估其作為化療增敏劑之潛力與分子機制。本研究使用人類牙齦正常上皮細胞 (SG)及舌頭鱗狀上皮癌細胞株 (SAS)，透過Presto Blue分析評估細胞存活率；透過傷口癒合試驗與Transwell試驗檢測細胞遷移能力；藉由醛脫氫酶試驗分析癌幹性；利用流式細胞儀檢測活性氧類並分析細胞凋亡以探討細胞死亡機制。最後以西方墨點法分析關鍵蛋白表現。結果顯示，檸檬醛對正常上皮細胞的毒性顯著低於SAS細胞，且合併順鉑能抑制SAS細胞遷移能力及上皮間質轉化 (epithelial-to-mesenchymal transition, EMT)過程，顯著增強SAS 細胞上皮標記物E-鈣黏蛋白 (epithelial cadherin, E-cadherin)，下調間質標記物波形蛋白 (Vimentin)、基質金屬蛋白酶2 (matrix metalloproteinase 2, MMP-2)與上皮間質轉化轉錄因子Twist相關蛋白 (twist-related protein, Twist)的蛋白質表現量。此外，檸檬醛亦降低SAS細胞的癌幹性指標，包含降低醛脫氫酶活性及癌幹性標記物NANOG蛋白 (homeobox transcription factor nanog, NANOG)與八聚體結合轉錄因子4 (octamer-binding transcription factor 4, OCT-4)的蛋白質表現量。機制上，檸檬醛合併順鉑會誘導SAS細胞大幅累積細胞內活性氧類 (reactive oxygen species, ROS)，顯著活化c-Jun胺基末端激酶 (c-Jun NH2-terminal kinase, JNK)/胱天蛋白酶3 (caspase-3, Casp3)凋亡信號通路，提升處於早期凋亡的細胞比例。總結而言，在不增加對正常細胞毒性的前提下，此合併療法能透過雙重機制發揮協同抗癌作用：其一，抑制遷移並削弱癌幹性；其二，促進ROS產生並誘導細凋亡。本研究成果為開發針對口腔癌之合併治療策略，提供強而有力的實驗依據與極具潛力的候選藥物組合。

Oral cancer is highly aggressive, presenting a poor prognosis and high mortality rates due to the difficulty of early diagnosis and the limited efficacy of existing treatment strategies. Cisplatin, a commonly used chemotherapeutic agent for oral cancer, is limited by its side effects and the development of drug resistance in cancer cells. Recent research has drawn significant attention to the natural compound citral, which has demonstrated anti-cancer properties and tumor selectivity across various cancers. However, studies on citral in the context of oral cancer have been quite limited to date. Therefore, this study aimed to investigate whether citral can inhibit the migration of oral cancer cells and to evaluate its potential as a chemosensitizer and the underlying molecular mechanisms when combined with cisplatin. Specifically, we used human normal gingival epithelial cells (SG) and tongue squamous cell carcinoma cells (SAS) to conduct the following experiments. Cell viability was assessed by Presto Blue assay; cell migration was examined using wound-healing and Transwell assays; cancer stemness was analyzed aldehyde dehydrogenase (ALDH) activity assay and reactive oxygen species (ROS) levels and apoptosis were detected via flow cytometry. Finally, the expression of key proteins was analyzed by Western blot. The results showed that citral exhibited significantly lower toxicity to normal epithelial cells compared to SAS cells. The combination of citral and cisplatin effectively suppressed SAS cell migration and the epithelial-to-mesenchymal transition (EMT) process by downregulating the protein expression of the epithelial marker E-cadherin while downregulating the protein expression of mesenchymal markers Vimentin, MMP-2, and the EMT-transcription factor Twist in SAS cells. Furthermore, citral also reduced cancer stemness, indicated by decreased ALDH activity and lower protein expression of NANOG and OCT-4. Mechanistically, the combined treatment induced a substantial accumulation of intracellular ROS in SAS cells, significantly activated the JNK/caspase-3 apoptotic signaling pathway, and increased the proportion of cells undergoing early apoptosis. In conclusion, without increasing cytotoxicity to normal cells, this combination therapy exerts synergistic anti-cancer effects through a dual mechanism: (1) inhibiting migration and attenuating cancer stemness, and (2) promoting ROS production and inducing apoptosis. The findings of this study provide strong experimental evidence and a highly promising candidate drug combination for developing novel combined therapeutic strategies against oral cancer.