牡丹皮抑制腎癌細胞的轉移與增生

Abstract

腎細胞癌在2007年為全世界發生率第七位的癌症，也是最常見的腎臟惡性腫瘤。而在人類惡性腫瘤中，腎細胞癌佔3 %且每年影響全世界約150,000人，造成約78,000人死亡。大部分的腎細胞癌常常出現血管新生以及細胞入侵能力的提升，加上對傳統治療具有抗性，因此針對有抗性的腎細胞癌患者，近年來細胞激素治療，包含干擾素-α (IFN-α) 與介白素-1，-2，-12 (IL-1,-2,-12) 被大量發展，美國食品暨藥物管制局也允許應用於高度轉移性腎細胞癌已快二十年。然而，細胞激素的使用在中等風險的病人中仍然受限，因此針對於治療腎細胞癌，新藥物的開發與研究應要持續進行。 由於許多國家中的癌症病人開始接受中藥搭配或取代傳統化學治療方式，因此我們以786O細胞株來檢測許多中藥在抑制細胞增生與細胞轉移的效果。我們發現相較於其他中藥，牡丹皮（Paeonia suffruticosa, PS）在細胞實驗與動物實驗中都顯著地抑制細胞轉移與增生，牡丹皮源自牡丹花的樹皮並在過去被應用於許多疾病上，例如：斑、癲癇、經期不規律等。而在過去幾十年的科學研究上，牡丹皮已經被證明有抗增生、抗發炎、抗糖尿病、神經保護等功能。 在本篇研究中，為了探究牡丹皮如何達到抑制細轉移與增生的效果。我們先以即時定量聚合酶連鎖反應 (Quantitative Real-time PCR) 與西方點墨法 (Western blot) 找出被牡丹皮影響的轉移相關基因。我們證明牡丹皮抑制N-cadherin表現量。這樣的抑制效果可能是透過細胞核內β-catenin減少而使Slug表現量下降加上Slug轉置至到細胞核的量也減少；除此之外，我們還發現牡丹皮會影響VEGFR-3/FAK/PI3K訊息傳導路徑來抑制Rac-1的活化而導致肌動蛋白絲形成減少，造成細胞轉移或移動能力降低。 除了抗轉移能力之外，牡丹皮也有良好的抗增生能力，可抑制cyclin A使細胞停滯於細胞周期中的S期，另外，也可以發現Bcl-2減少以及PARP切割增加的情形，代表牡丹皮也可促進細胞凋亡。牡丹皮的抗增生效果可藉由搭配cisplatin與adriamycin來達到協同作用；牡丹皮與cisplatin的協同作用範圍坐落在高抑制區，更具有臨床上的應用價值。根據以上的實驗結果，我們證明牡丹皮同時擁有抑制轉移與增生的能力，亦可以與傳統化學治療一起使用達到協同效果，在未來腎細胞癌的治療上，牡丹皮具有相當大的發展潛力。

Renal cell carcinoma (RCC) is responsible for the majority tumor malignancies arising in the kidney, making it the 7th most common cancer among the world in 2007. It accounts for 3 % human malignancies and influences almost 150,000 people worldwide each year, causing approximately 78,000 deaths annually. Most cases of RCC appear to occur sporadically which accompany increased angiogenesis and invasive ability. Moreover, RCC is resistant to conventional therapies, including radiation and chemotherapy. Aimed at the patients with no response to conventional therapy, the cytokine-based therapies including interferon-α (IFN-α), and interleukin-1,-2, and -12 (IL-1,-2, and -12) have been developed and further approved for the treatment of metastatic RCC by US FDA for almost 20 years. However, the effect of cytokines is very limited in intermediate-risk patients. Therefore, development of new agent which inhibits tumor metastasis is crucial for treatment of RCC. As CHMs is identified as one of alternative strategies in the treatment of many cancers among many countries, we examine the effects of various CHMs on proliferation and migration of 786O cells by MTT assay and migration assay. We found that Paeonia suffruticosa (PS) exhibits stronger anti-metastasis and anti-proliferation activities than other CHMs in vitro and in vivo. Paeonia suffruticosa (PS) has been used in the treatment of various diseases such as macula, epilepsy, and menstrual disorders in tradition prescriptions. In our study, we performed quantitative real-time PCR and western blot analysis to dissect mechanisms affected by PS. Our finding indicates that PS suppresses N-cadherin expression. Through decreased phosphorylation of Akt and Src, noncanonical phosphorylation of β-catenin causes its localization from cytoplasm to nucleus and further triggers Wnt-β-catenin target genes including Slug. Reduction of Slug impairs the transcription of N-cadherin. It is demonstrated that PS suppresses the activation of Rac-1 via VEGFR-3/FAK/PI3K pathway, leading to the decreased formation of stress fiber and inhibition of migration. Additionally, PS induces intrinsic apoptosis pathway and cause S phase arrest. Consequently, we provided the potential remedy that combination of PS with cisplatin or adriamycin, respectively, could synergistically enhance the cytotocicity of RCC in a different manner. In particular, PS and cisplatin in combination have synergistic effects in high inhibitory range. These results described above suggest that PS has dual effects on migration and proliferation and can be administrated to the patients with RCC.