褪黑激素合併福退癌誘發腎臟癌細胞粒線體自噬凋亡分子機轉研究

腎細胞癌是最常見腎癌種類，由於腎臟為沉默器官，且因其具標靶藥物抗藥性，導致腎細胞癌難以治癒。福退癌 (pazopanib) 是一種口服型多重酪氨酸激酶抑制劑，為腎癌標靶藥物。褪黑激素是一種人體內生性荷爾蒙，功能為調節日夜生理節奏，近年來已發現褪黑激素對於多種癌症抗癌效果良好，然其合併標靶藥物治療機制仍未清楚。因此，本研究主軸為探討褪黑激素合併腎癌標靶藥物福退癌在治療腎癌之詳細機制。首先，我們發現褪黑激素引起腎細胞癌凋亡。接著，探討福退癌結合褪黑激素治療腎癌細胞效果，結果顯示合併療法更有效抑制細胞生長，細胞凋亡比率、粒線體膜電位改變比率大幅提升，西方墨點法數據也一致。為了探究凋亡上游訊息傳遞路徑，西方墨點法發現p38蛋白活化。透過酸性胞器染色螢光變強與西方墨點法之自噬標誌蛋白LC3活化，確立合併療法同時引起凋亡與自噬。為了探究凋亡、自噬關聯性，使用p38抑制劑與siRNA-siLC3等自噬抑制劑相互驗證。結果顯示，若p38活性抑制時，腎癌細胞凋亡與自噬作用皆減弱，而當自噬遭到抑制時，發現腎癌細胞大量死亡，推論此為保護型自噬作用。為了深入探討自噬機制，藥物處理下PTEN-induced kinase 1 (PINK)/Parkin、BCL2 Interacting Protein 3 (BNIP3)表現上升，透過粒線體自噬螢光染劑、西方墨點法發現粒線體自噬引發。除此之外，探討合併療法是否影響癌幹細胞生理機制，發現癌幹細胞生長、癌幹細胞標誌蛋白皆被抑制。最後，動物實驗發現，合併療法有效抑制腎細胞癌生長。綜合以上，合併療法透過p38/MAPK活化引發粒線體-自噬型細胞凋亡，藉此抑制腎癌細胞生長，此外，合併療法亦可抑制腎癌幹細胞生長。總結來說，本篇研究的合併療法可能成為腎癌治療潛在方法，為癌症治療注入新血。

Renal cell carcinoma (RCC), the most fatal of all urological malignancies, is the most common renal cancer. Because of the resistance to target therapy, the mortality rate of RCC keeps high despite the improved treatment. Pazopanib, an orally available multi-TKI, has been approved for the treatment of RCC. Melatonin, a hormone regulates the day-night cycles, has been shown the effect of anti-metastasis in RCC cell lines on previous study, but another anti-tumor mechanism in RCC with melatonin treatment has not been reported. Here, we explore the effect of combination treatment with melatonin and pazopanib in RCC cell lines. First, we explored the effect of melatonin in RCC cells, and the results revealed that melatonin inhibited the RCC cell proliferation via induction of apoptosis. To demonstrate the effect of combination treatment of melatonin and pazopanib on RCC cells, our results showed that cellular viability was effectively decreased via combination treatment. Combination treatment strongly enhanced apoptosis and the change of mitochondrial membrane potential when compared to melatonin treatment alone. Furthermore, combination treatment induced p38-mediated apoptosis of RCC cells. Additionally, the data of acidic vesicular organelles (AVO) stain demonstrated that combination treatment caused autophagy, and also induced the autophagy biomarker, LC3II. To explore the relationship between apoptosis and autophagy, the p38 inhibitor, BIRB-796, LC3 siRNA and autophagy inhibitors were used. The results displayed that combination treatment-induced apoptosis and autophagy were suppressed by BIRB-796; however, combination treatment-induced apoptosis was promoted after siLC3 transfection, suggesting that the induction of autophagy was used to protect RCC cells from combination therapy-induced apoptosis. In addition, we delved into the mechanism of autophagy induction, and the results of immunofluorescence staining by using the mitophagy detection kit revealed that mitophagy was induced through activation of PINK/Parkin and BNIP3 pathway and LC3 on mitochondria. Also, we found that combination therapy inhibited the stemness of RCC via inhibition of cancer stem cell (CSC) biomarkers. Results from in vivo animal model were similar to the results from in vitro cell experiments. In this study, our results showed that pazopanib effectively promotes melatonin-enhanced autophagic apoptosis through activation of p38/MAPK pathway in RCC cell lines. In conclusion, the combinatorial strategy may serve as the potential treatment in renal cell carcinoma