5-去甲基川陳皮素透過抑制HIF-1α/CAIX與AKT磷酸化軸向阻斷子宮頸癌轉移之分子機制

子宮頸癌是全球女性常見惡性腫瘤之一。雖然子宮頸抹片篩檢與人類乳突病毒（human papillomavirus, HPV）疫苗接種已有效降低子宮頸癌發生率與死亡率，但在醫療資源不足、篩檢普及率較低及疫苗覆蓋率有限的地區，子宮頸癌仍造成沉重疾病負擔。此外，部分患者在診斷時已進展至晚期，或在治療後發生復發與遠端轉移，使臨床治療仍面臨挑戰。因此，尋找具抗腫瘤與抗轉移潛力的新型候選化合物，仍是子宮頸癌研究的重要方向。多甲氧基黃酮類化合物廣泛存在於柑橘類植物中，具有抗發炎、抗氧化與抗癌潛力。5-去甲基川陳皮素（5-Demethylnobiletin, 5DMN）與3-去甲基川陳皮素（3-Demethylnobiletin, 3DMN）為臺灣香檸（Citrus depressa）中分離出的多甲氧基黃酮；然而，其在子宮頸癌細胞遷移、侵襲及相關分子機制中的作用仍未明確。 本研究比較5DMN與3DMN在子宮頸癌細胞中的抗腫瘤潛力，並進一步解析5DMN抑制細胞遷移與侵襲的分子機制。MTT分析顯示，5DMN可降低HeLa與SiHa細胞存活率，而3DMN對子宮頸癌細胞影響較不明顯，因此後續研究聚焦於5DMN。在中低濃度處理下，5DMN未明顯誘導細胞週期停滯，但已能抑制HeLa與SiHa細胞遷移及侵襲能力，顯示其抗轉移作用並非單純來自細胞毒性。RNA-seq分析顯示，碳酸酐酶IX（carbonic anhydrase IX, CAIX）可能為5DMN調控子宮頸癌細胞轉移能力的重要標的。後續qRT-PCR與Western blot驗證確認，5DMN可抑制CAIX mRNA與蛋白表現；CAIX過度表現與基因沉默實驗也進一步支持CAIX參與5DMN調控細胞遷移與侵襲的過程。機制分析顯示，5DMN可抑制AKT磷酸化，並降低HIF-1α核轉位與轉錄活性。Wortmannin合併處理、CoCl₂活化HIF-1α及ChIP-qPCR分析進一步指出，5DMN可能透過AKT/HIF-1α路徑，降低HIF-1α在CAIX啟動子區域的結合能力，進而抑制CAIX表現。體內實驗結果顯示，口服給予5DMN可減少子宮頸癌遠端轉移模型中的肺部腫瘤結節形成，並伴隨腫瘤組織中CAIX與Ki-67表現下降；主要器官H&E染色則未觀察到明顯組織病理變化。 綜合以上結果，本研究證實5DMN可透過抑制AKT磷酸化，降低HIF-1α核轉位及其對CAIX啟動子區域的結合能力，進而降低CAIX表現，抑制子宮頸癌細胞遷移、侵襲與體內遠端轉移。本研究建立5DMN調控AKT/HIF-1α/CAIX訊號軸的分子機制，也提供其作為子宮頸癌抗轉移候選天然化合物的實驗依據與後續轉譯研究基礎。

Cervical cancer remains one of the most common malignancies among women worldwide. Although Pap smear screening and human papillomavirus (HPV) vaccination have reduced its incidence and mortality, cervical cancer remains a significant health burden, particularly in regions with limited medical resources and inadequate screening and vaccination coverage. Moreover, advanced disease, recurrence, and distant metastasis remain major clinical challenges. Therefore, identifying novel compounds with anti-tumor and anti-metastatic potential is important. 5-Demethylnobiletin (5DMN) and 3-demethylnobiletin (3DMN) are polymethoxyflavones isolated from Citrus depressa; however, their effects on cervical cancer progression remain unclear.In this study, we compared the anti-tumor effects of 5DMN and 3DMN in cervical cancer cells and investigated the molecular mechanisms underlying the anti-metastatic activity of 5DMN. MTT assays showed that 5DMN reduced the viability of HeLa and SiHa cells, whereas 3DMN exhibited limited effects. At low to moderate concentrations, 5DMN did not markedly induce cell cycle arrest but significantly suppressed cervical cancer cell migration and invasion, suggesting that these effects were not solely attributable to cytotoxicity. RNA-seq analysis identified carbonic anhydrase IX (CAIX) as a potential target of 5DMN. qRT-PCR and Western blot analyses confirmed that 5DMN reduced CAIX mRNA and protein expression, while CAIX overexpression and gene-silencing experiments further supported its involvement in 5DMN-mediated inhibition of cell migration and invasion. Mechanistically, 5DMN inhibited AKT phosphorylation and reduced HIF-1α nuclear translocation and transcriptional activity. Wortmannin treatment, CoCl₂-mediated HIF-1α activation, and ChIP-qPCR analysis further demonstrated that 5DMN suppressed CAIX expression through the AKT/HIF-1α pathway by reducing HIF-1α binding to the CAIX promoter. In vivo, oral administration of 5DMN reduced lung tumor nodule formation in a cervical cancer distant metastasis model and decreased CAIX and Ki-67 expression in tumor tissues, with no obvious histopathological changes in major organs.Taken together, these findings demonstrate that 5DMN suppresses cervical cancer cell migration, invasion, and distant metastasis by inhibiting the AKT/HIF-1α/CAIX signaling axis. This study provides mechanistic and experimental evidence supporting 5DMN as a potential natural compound for the development of anti-metastatic strategies against cervical cancer.