甘草查爾酮A透過自噬作用抑制腎臟癌細胞移動和轉移作用

腎臟細胞癌 (RCC)是一種常見且惡性的腎癌類型，且RCC患者時常會發生遠處轉移。甘草查爾酮A (Licochalcone A)是一種從甘草根中分離得到的天然查爾酮，具有抗腫瘤、抗發炎及抗血管新生等多種生物功能。然而，甘草查爾酮A抗腎臟癌作用機制仍不清楚。使用MTT方式檢測甘草查爾酮A處理的786-O及Achn細胞存活率，MTT結果發現隨著甘草查爾酮A濃度及時間增加會抑制786-O和 Achn細胞存活率，使用LDH方法、PI染色及細胞凋亡實驗證實不會產生細胞毒性和細胞凋亡。另外，我們發現甘草查爾酮A會顯著抑制786-O及Achn細胞的轉移能力及誘導自噬指標性蛋白 (LC3β)及p38磷酸化增加，利用抑制LC3β (siRNA-LC3β)或是抑制p38 (si-p38)方式證實會減少甘草查爾酮A抑制細胞轉移能力及LC3β的表現。進一步使用PROMO軟體預測Sp1可能調控LC3β基因的轉錄作用，實驗證實甘草查爾酮A抑制Sp1的表現，反之，過度表現Sp1會減少甘草查爾酮A抑制細胞轉移能力。另一方面，我們也發現甘草查爾酮A會減少FAK及Src磷酸化表現，且進一步使用PF-753228 (FAK抑制劑)及PP-2 (Src抑制劑)來證實FAK與Src的活化參與甘草查爾酮A抑制腎臟癌細胞轉移過程。最後我們也結合了臨床抗癌藥sorafenib與甘草查爾酮A合併使用在786-O及Achn細胞中，結果發現合併使用能夠更顯著抑制腎臟癌細胞的移動和轉移。綜合以上研究證實，甘草查爾酮A透過細胞自噬作用來抑制腎臟癌細胞的轉移作用，期待甘草查爾酮A成為新穎的天然抗癌藥物，應用於臨床腎臟細胞癌轉移的治療。

Renal Cell Carcinoma (RCC) is the malignant, common type of kidney cancer and patients with RCC often develop distant metastasis. Licochalcone A (Lico A) is a natural chalcone isolated from the root of Glycyrrhiza uralensis that possesses various biological functions including anti-tumor, anti-inflammatory, and anti-angiogenesis effects. However, the mechanism underlying the antitumor effects of Lico A in RCC remains unclearly. Cell viability of 786-O and Achn treated with LicoA was measured by MTT assay. The result found that Lico A treatment for 24 hours had no significant effect on 786-O, but Lico A in Achn treated 25 and 31.25 μM reduced the cell survival rate to about 80%. Therefore, we used the LDH-Cytotoxicity Colorimetric Assay to detect Lico A treated 786-O and Achn for 24 hours, and found that Lico A treatment did not produce cytotoxicity. Then we analyzed whether Lico A in RCC cells would affect the cell cycle and apoptosis, and found that there was no significant effect. In addition, Lico A inhibits the migration and invasion ability of 786-O and Achn cells. Previous researches suggested that autophagy might involve in metastatic ability, we found that Lico A induced autophagy and increased the expression of the autophagy markers (LC3β) in 786-O and Achn cells. Moreover, transfection with siRNA-LC3βin Lico A-treated cells to found significantly restored the ability of cell migration and invasion. Then use PROMO to predict the promoter of the LC3β gene. It was found that Sp1 may bind to it, and Lico A would reduce the expression of Sp1. In addition, we found that Lico A can increase the expression of p-p38, and it is related to the inhibition metastasis of RCC cells by Lico A. On the other hand, we also found that Lico A can reduce p-FAK and p-Src in 786-O and Achn, and further use PF-753228 (FAK inhibitor) and PP-2 (Src Inhibitor) to confirm that FAK and Src are involved in the process of Lico A inhibiting migration and invasion of RCC cells. Finally, we also combined the clinical anticancer drug sorafenib and Lico A in 786-O and Achn, and found that the combined use can more significantly inhibit cell migration and invasion. In conclusion, it is confirmed that Lico A can inhibit the migration and invasion of RCC cells through autophagy. It is expected that Lico A will become a novel natural anticancer drug and be used in the treatment of renal cell carcinoma metastasis.