紫草素促進腎癌細胞死亡的分子機轉

腎臟細胞癌（renal cell carcinoma, RCC）是泌尿系統最常見的惡性腫瘤之一，約佔成人惡性腫瘤的3％，且全球發病率依舊在逐年上升。大多數的 RCC 患者在接受細胞毒性或標靶藥物治療期間都會有抗藥性產生，所以急需更進一步的研究來從天然物質中找出更有效且較無毒性的抗癌活性成份治療腎臟癌。紫草素 (shikonin) 是一種天然的萘醌類 (naphthoquinone)，主要存在於中國傳統中草藥材紫草 (Lithospermum erythrorhizon) 根莖部，具有抗癌、抗發炎及促進傷口愈合的作用，但目前紫草素對於腎癌細胞的影響尚未清楚。本研究主要探討紫草素對於腎臟癌細胞之抗癌效果以及其相關分子機轉。首先，以 MTT 測試紫草素對腎臟癌細胞 (ACHN 和 CAKI-1) 的毒殺效果，發現隨著紫草素濃度 (4、6、8 μM) 增加，細胞存活率明顯被抑制。進一步利用螢光染劑 DCFH-DA 偵測出紫草素會促進細胞內活性氧化物大量增加；再使用 JC-1 染劑測其粒腺體膜電位也可清楚觀察到紫草素會造成粒腺體膜電位下降的趨勢。另外透過 annexin V/PI 雙重染色法發現紫草素能造成 ACHN 及 CAKI-1 同時引起凋亡與壞死現象。然而預先處理抗氧化劑如 N-acetyl cysteine (NAC) 再加入紫草素發現無論是細胞存活率、細胞內活性氧化物質、粒腺體膜電位以及細胞死亡現象皆有恢復的情況。進一步以 Western blot 分析發現紫草素可誘導凋亡相關蛋白 pro-caspase 3 及 PARP 被切割活化，BAX 則無明顯改變，預先處理 NAC 可恢復蛋白表現量。接著分析 MAPK 相關蛋白發現磷酸化 ERK 1/2 蛋白表現量會隨著紫草素處理濃度增加而逐漸下降，磷酸化 P38 則會明顯上升；而預先處理 NAC 的組別磷酸化 ERK 1/2 蛋白表現量會回升，磷酸化 P38 則會明顯下降，證實紫草素可透過 ERK 1/2 與 P38 訊息傳遞路徑調控細胞凋亡。另外 Western blot 分析也發現自噬相關蛋白 LC3-II 表現量大幅度增加，同樣在預先處理 NAC 的組別 LC3-II 蛋白則無表現。綜合以上結果顯示，紫草素藉由提高細胞內活性氧化物抑制腎癌細胞生長，造成細胞死亡現象發生。

Renal cell carcinoma (RCC) is represented as one of the most common malignancy tumours in adults and its global incidence continues to rise annually. Most of the RCC patients often show unsatisfactory outcomes and typically develop drug resistance after being treated with cytotoxic drugs or targeted therapy. Hence, it is critical to identify novel and effective therapeutic drugs with less toxicity to improve the clinical outcomes of RCC. Shikonin is a natural red naphthoquinone compound extracted from the dried root of traditional Chinese herbal medicine Lithospermum erythrorhizon. Recently, scattering reports showed that shikonin possesses various beneficial functions including anticancer, anti-inflammatory and wound-healing effects. To date, the biological function of shikonin in RCC still remains unclear. This research aims to investigate the mechanism underlying the anticancer effects of shikonin in RCC. The results of MTT assay demonstrated that shikonin significantly reduced the cell viability of ACHN and CAKI-1 cells in a dose-dependent manner. The effects of shikonin on cell proliferation could be recovered with antioxidant reagents pre-treatment such as N-acetyl cysteine (NAC). In addition, DCFH-DA staining analysis also detected the elevation of the cellular concentration of reactive oxygen species (ROS) in response to shikonin treatment, suggesting the inhibitory effects of shikonin in ACHN and CAKI-1 through the generation of ROS. Furthermore, alterations in mitochondrial membrane potential (MMP) were confirmed by flow cytometry using JC-1 dye in shikonin-treated groups. Western blot analysis revealed that shikonin enhanced the activation of apoptosis-related proteins such as pro-caspase 3 and PARP whereas the protein expression of BAX remains unchanged. Shikonin also significantly affected the expression level of MAPK proteins, led to an increase of p38 phosphorylation level but decreased in ERK phosphorylation. Besides, the expression of autophagy marker LC3-II was significantly increased in response to shikonin treatment. These phenomena could be rescued by pretreatment of NAC. These results indicated that shikonin could cause cell death of RCC cells via generation of ROS, suggesting that it could be developed as a potential anti-cancer agent.