謝逸憲邱慧玲HSIEH, YI-HSIENCHIOU, HUI-LING蔡辰憶TSAI, CHEN-YICHEN-YITSAI醫學檢驗暨生物技術學系碩士班2026-10-162026-07-3020262026-07-16https://handle.ncl.edu.tw/11296/m5gzckhttps://scholars.csmu.edu.tw/handle/123456789/22196骨肉瘤 (osteosarcoma, OS) 為兒童與青少年中最常見之原發性惡性骨腫瘤，具有高度侵襲性與早期轉移之特性，臨床治療成效仍有待提升，因此尋找具潛力之新穎治療策略相當重要。Thonningianin A (ThA) 為一種存在於植物中的天然鞣花單寧類黃酮化合物，已被證實具有抗氧化、抗發炎及抗腫瘤等生物活性，然而在骨肉癌中的抗癌作用及分子調控機制尚未釐清。研究結果顯示 ThA 能顯著抑制骨肉癌細胞株之細胞增殖與群落的形成能力；同時，亦可誘導細胞活性氧的產生與累積，進而促使粒線體功能障礙與細胞凋亡。在分子層面上，能觀察到細胞自噬與凋亡之相關蛋白的表現；而抗氧化劑 (NAC) 或siNIX、siBNIP3 則可減緩細胞自噬與凋亡的反應，證實ROS誘導粒線體自噬在 ThA 誘導骨肉癌細胞死亡中扮演相當重要的角色。 RNA 定序結果顯示，ThA 能顯著抑制致癌基因SKP2的表現，此結果亦透過 qPCR 與西方墨點法證實。為了尋找 SKP2的上游調控路徑，我們發現 ThA 可專一性地促使 JNK 磷酸化，但對 ERK 和 p38 的磷酸化則無影響。進一步實驗證實，使用 JNK 抑制劑可逆轉 ThA 引起的生長抑制、細胞自噬與凋亡，並且恢復 SKP2 的表現；而使用 NAC 處理也能恢復 SKP2 的表達，顯示 ThA 是透過 ROS/p-JNK 路徑進而減少 SKP2 的表現。此外，ThA 與化療藥物 Cisplatin 併用時，對骨肉癌細胞生長具有協同抑制的效果，並有效抑制 SKP2 的表現，亦進一步增強 ROS 所誘導的細胞自噬與凋亡作用。本研究證實 ThA 誘導活性氧分子並透過 p-JNK/SKP2 路徑來促進骨肉癌細胞自噬與凋亡，進而有效抑制骨肉癌的進展，並具有作為骨肉癌輔助治療天然化合物之潛在應用價值。Osteosarcoma (OS) is the most common primary malignant bone tumor in children and adolescents and is characterized by high invasiveness and a strong propensity for early metastasis. ThA, a naturally occurring ellagitannin flavonoid found in plants, has been reported to exhibit antioxidant, anti-inflammatory, and antitumor activities in various cancer types. However, the effects and underlying molecular mechanisms of ThA in osteosarcoma remain largely unexplored. Experimental results demonstrate that ThA significantly inhibits the proliferation and colony formation capabilities of osteosarcoma cell lines. Concurrently, it induces the generation and accumulation of intracellular ROS, thereby triggering mitochondrial dysfunction and apoptosis. At the molecular level, altered expressions of proteins related to autophagy and apoptosis were observed. Furthermore, treatment with the antioxidant NAC or knockdown of NIX/BNIP3 attenuated these autophagic and apoptotic responses, confirming that ROS-mediated mitophagy plays a critical role in ThA-induced osteosarcoma cell death. RNA sequencing analysis revealed that ThA significantly suppresses the expression of the oncogene SKP2, a finding further validated by qPCR and Western blotting. In searching for the upstream regulatory pathway of SKP2, we discovered that ThA specifically promotes JNK phosphorylation while exerts no effect on the phosphorylation of ERK and p38. Further experiments confirmed that pharmacological inhibition of JNK reverses ThA-induced growth inhibition, autophagy, and apoptosis, while restoring SKP2 expression. Additionally, treatment with NAC also rescued SKP2 expression, indicating that ThA downregulates SKP2 via the ROS/p-JNK pathway. Moreover, the combination of ThA and the chemotherapeutic drug Cisplatin exhibits a synergistic inhibitory effect on osteosarcoma cell growth, effectively suppressing SKP2 expression and further amplifying ROS-induced autophagy and apoptosis. This study demonstrates that ThA induces ROS generation and promotes autophagy and apoptosis in osteosarcoma cells through the p-JNK/SKP2 pathway, thereby effectively inhibiting osteosarcoma progression. These findings suggest that ThA possesses potential therapeutic value as a natural compound for adjuvant osteosarcoma treatment.骨肉癌趕黃草苷ASKP2細胞自噬細胞凋亡ROSOsteosarcomaThonningianin AS-phase kinase-associated protein 2autophagyapoptosisthesis