肉桂醛誘導人類骨肉瘤細胞程序性死亡路徑之研究

骨肉瘤(Osteosarcoma, OS)為骨癌的一種，為最常見的原發性惡性骨腫瘤，好發於兒童及青少年，罹患該腫瘤之病患通常預後差、死亡率高，且深陷現有化療藥物所引起的強副作用之中，病患迫切需要新的安全有效之藥物進行治療，因此，從天然物為來源所開發的抗癌藥物備受關注。肉桂醛，為一種從中草藥-肉桂中，所分離出來的化合物，已被證實具有有效之抗菌、抗炎及抗癌……等特性。然而，肉桂醛對於骨肉瘤的影響尚未得到充分的研究，因此，本研究探討肉桂醛對骨肉瘤細胞的抗癌作用及其潛在的機制。本實驗將骨肉瘤細胞株143 B、HOS處理肉桂醛，接著利用MTT測定和集落形成測定來觀察細胞增殖和群落形成的能力，並進行細胞週期分析、DAPI染色法、酸性囊狀胞器染色分析法、粒線體膜電位測定和annexin V-FITC/PI染色法來分析肉桂醛對骨肉瘤細胞之凋亡與自噬的影響。接著，以西方墨點法(western blotting)進一步探究其可能的潛在機制。研究結果顯示，肉桂醛會透過粒線體訊息傳遞路徑誘導骨肉瘤細胞進行程序性死亡，並抑制細胞增殖和群落形成。此外，透過螢光顯微鏡和流式細胞術相關實驗也顯示骨肉瘤細胞經肉桂醛處理後，誘發染色質產生聚集、粒線體膜電位去極化，造成細胞凋亡，同時，細胞之酸性囊狀胞器增加，顯示肉桂醛誘導細胞自噬。繼續深入進行western blotting實驗發現，凋亡抑制相關蛋白Bcl-2的蛋白表現量及其上游促進細胞存活相關蛋白之磷酸化FAK、Akt、mTOR的蛋白表現量均顯著下降，而凋亡促進蛋白cytochrome c的蛋白量表現量顯著性地增加。總結，本研究證實肉桂醛可抑制人類骨肉瘤細胞的生長與增殖，並透過粒線體訊息傳遞路徑與抑制FAK/Akt/mTOR訊息傳遞路徑來誘導人類骨肉瘤細胞的程序性細胞死亡。

Osteosarcoma (OS) is the most common primary bone tumor with poor survival that primarily affects children and adolescents. Taming osteosarcoma requires urgent search for some new efficient methods with safety for clinical therapy. Therefore, there has been an interest in the development of anticancer drugs from natural sources, and cinnamaldehyde is an example. Cinnamaldehyde, a compound isolated from the Chinese herb cinnamon that has been shown to possess substantial anticancer properties. However, the anticancer activities effect of cinnamaldehyde against OS has never been investigated. This study systematically evaluates the anticancer activities effects of cinnamaldehyde on Osteosarcoma cells. OS cells were incubated with increasing doses of cinnamaldehyde (0, 20, 40, 60 μM) for 24 hours. Then, the cell proliferation and colony formation was determined by MTT assay and colony formation assay. The apoptotic and autophagy effects of cinnamaldehyde on OS cells was determined by PI stain, DAPI Staining, AVOs stain, mitochondrial membrane potential assay and annexin V- FITC/PI stain. Then, its possible underlying mechanisms were further studied by using western blotting analysis. Studies suggested that cinnamaldehyde via mitochondrial signaling pathway induces the programmed cell death of OS. Cells treated with cinnamaldehyde, it suppressed proliferation and colony formation. Moreover, the data showed the phenomenon of chromatin aggregation, depolarization of the transmembrane potential, development of acidic vesicular organelles and phenomenon of apoptosis by fluorescence microscopy and flow cytometry. There was a reduction in Bcl-2 expression and FAK, Akt, mTOR phosphorylation. Furthermore, there was a upregulation in expression of cytochrome c. Together, these data suggest that cinnamaldehyde treatment suppresses OS growth and induces the programmed cell death of OS via mitochondrial signaling pathway and FAK/Akt/mTOR inhibition signaling pathway.