| 摘要: | 結直腸癌 (Colorectal cancer, CRC)是全球常見且死亡率高的惡性癌症，且患病率隨著年齡增長而上升，手術是主要治療方法，搭配術後化療可以減少復發機會，但癌細胞可能產生轉移或復發，因此，深入探討化療藥物在癌細胞內的作用機制，並開發新型藥物以提升治療效果，具有重要臨床意義。多種癌細胞具有對Nuclear factor erythroid 2-related factor 2 (Nrf2)「成癮」現象，其過度活化會促使癌細胞產生抗藥性，進而降低化療效果。順鉑 (Cisplatin)雖為常用第一線化療藥物，但其治療效果易受到抗藥性的影響。Nrf2 訊息路徑與細胞自噬機制，常與癌症幹細胞 (cancer stem cells, CSC)產生及抗藥性密切有關。本研究延續先前實驗室研究成果，選用硒化合物-左旋硒半胱胺酸 (L-selenocystine, SeC)，其可有效抑制Nrf2訊息路徑及細胞代謝相關的自噬功能，且對正常細胞則無明顯細胞毒性。為進一步提升藥物穩定性與傳送效率，本研究論文利用新開發含硒奈米金藥物 (Au@SeC)，探討其對Nrf2成癮以及Nrf2成癮與順鉑抗藥性的結直腸癌細胞的治療潛力。研究結果顯示，Au@SeC對Nrf2成癮的人類結直腸癌細胞株WiDr具選擇性細胞毒性，對正常腸道上皮細胞則無顯著影響。Au@SeC可抑制細胞內抗氧化途徑(Nrf2/Keap1/p62)與自噬相關訊息途徑，其機制與未包覆藥物 (free drug)SeC相似。此外，本研究使用以順鉑長期處理所建立之抗藥性細胞株CSCL細胞進行分析，此細胞表現出的CSC 蛋白標誌、免疫逃脫相關蛋白 (PD-L1、COX-2)以及上皮-間質轉化(epithelial-mesenchymal transition, EMT)相關間質蛋白特性。Au@SeC可有效抑制CSCL細胞中上述蛋白質表現，並誘導粒線體氧化壓力與粒線體自噬 (Mitophagy)，推測其可能藉由干擾粒線體功能與溶酶體恆定性造成細胞死亡。同時，Au@SeC亦能引發內質網壓力以及免疫原性細胞死亡(immunogenic cell death, ICD)，進而促進巨噬細胞M1極化。此外，Au@SeC可以降低PD-L1的蛋白質表現量，有助於改善腫瘤微環境並減少抗藥性產生。綜合上述結果， Au@SeC具備抑制Nrf2與細胞自噬、誘導細胞死亡、干擾免疫逃脫及改善腫瘤微環境之多重功能，顯示其作為新型抗癌藥物之潛力，未來有望應用於針對Nrf2成癮與CSC特性之癌細胞的治療策略。;Colorectal cancer (CRC) is one of the most common malignant cancers in the world, with incidence increasing with age. The surgery combined with chemotherapy or radiotherapy is effective in early stage of CRC but poorly effective in patient of metastasis or postoperative recurrence. Understanding the mechanisms of the chemotherapeutic agents and developing novel strategies to overcome drug resistance are critical importance. Many cancer cells exhibit addition of nuclear factor erythroid 2-related factor 2 (Nrf2), leading to hyperaction of the Nrf2 signaling pathway and subsequent drug resistance. Cisplatin, a commonly used first-line chemotherapeutic agent in CRC treatment, is significantly comprises its therapeutic efficacy due to the emergence of drug resistance. Aberrant Nrf2 signaling pathway and autophagy are closely associated with drug resistance and the development of cancer stem cells (CSCs)-like cells. Our previous studies demonstrated that the L-selenocystine (SeC) selectively targets Nrf2-addicted cancer cells by inhibiting Nrf2 signaling and autophagy pathway, with low toxicity to normal cells. In this study, we employed L-selenocystine-containing gold nanodrug (Au@SeC) to improve drug stability and delivery efficiency. We investigate the anti-cancer effects of Au@SeC on Nrf2-addicted CRC and cisplatin-resistant CSC-like cells. The results show that Au@SeC had selective cytotoxicity against Nrf2-addicted CRC cell line WiDr, without affecting normal intestinal epithelial cells. Au@SeC inhibits the Nrf2/Keap1/p62 antioxidant pathway and autophagy-related signaling, similar to the effects of free drug SeC. Furthermore, in the cisplatin-resistant CRC cell line CSCL cells —characterized by elevated CSC markers, immune escape-related proteins (PD-L1 and COX-2), and epithelial-mesenchymal transition (EMT) markers—Au@SeC effectively downregulated these protein expressions. Importantly, treatment with Au@SeC led to a reduction in mitochondrial structural and functional proteins, increased mitochondrial oxidative stress, and activated mitophagy. Notably, Au@SeC also induced lysosomal dysfunction, potentially impairing mitophagic flux and resulting in the accumulation of defective mitochondria. Additionaly, Au@SeC triggered endoplasmic reticulum (ER) stress and immunogenic cell death (ICD), promoting M1 polarization of macrophages. Au@SeC downregulated the expression of cyclooxygenase-2 (COX-2) and programmed death-ligand 1 (PD-L1), suggesting modulating the tumor microenvironment of CRC. Collectively, our findings suggest that Au@SeC exhibits multiple anticancer effects, including inhibition of Nrf2 and autophagy pathways, induction of cancer cell death, modulation of immune responses, and suppression of CSC-like properties These findings support the potential of Au@SeC as a promising therapeutic agent for targeting Nrf2-addicted and drug resistance CRC cells. |
