探討天然皂苷重樓在致癌基因KRAS突變的人類大腸直腸癌細胞造成的合成致死效果

Abstract

大約30%的癌症是由於RAS基因突變而成。KRAS是RAS的一種，影響腫瘤的生長和擴散。在大腸直腸癌(Colorectal cancer , CRC)中有30-45%為KRAS突變，但目前未能有針對性治療，導致預後不良。重樓皂苷Formosanin C(FC)是從Rhizoma Paridis saponins (RPS)分離的有效化合物，對肺癌，肝癌和食管癌有抑制作用。本實驗為確定FC是否可以对KRAS基因突變的大腸直腸癌病人有益，使用HT-29 KRAS正常型和HT-29 KRAS突變體兩株細胞。MTT的結果表明，FC對於KRAS突變的細胞抑制效果較佳，可能是FC靶向除KRAS突變型之外的另外一個基因或途徑，增強CRC的細胞毒性，造成合成致死。研究發現KRAS突變細胞產生的自噬狀況较佳，且在抑制劑BAF的預處理的条件下自噬比例明顯下降，證明自噬有走完，但也發現此細胞自噬是保護型的。Annexin V/propidium iodide雙染的結果表明，FC對於KRAS突變細胞產生的凋亡比例較多，没有引起細胞壞死。在抑制細胞自噬之後，會增加更多的細胞凋亡比例。另外， KRAS突變的細胞中FC在48小時可以誘發粒腺體自噬，也是保護性的粒腺體自噬。但KRAS正常的細胞在48小時觀察不到粒腺體自噬，猜測是粒腺體自噬已經走完，因為在西方墨點法中已經沒有Parkin蛋白的表現。綜上所述，在大腸直腸癌細胞中，KRAS突變細胞可透過FC誘發的細胞凋亡引起合成致死，使用細胞自噬粒腺體自噬的抑制劑可加成其效果。Formosanin C (FC), a component of “Yunnan Bai Yao”, is an anti-cancer compound isolated from Rhizoma Paridis saponins. Colorectal cancer (CRC) is the third most common cause of cancer-related death in Taiwan. The KRAS mutation in colorectal cancers accounts for about 35-45%. KRAS, a RAS isoform, affects the growth and spread of tumor. KRAS mutation failed to target therapies and lead to poor prognosis. To determine whether FC can enhance cytotoxicity of CRC with KRAS mutation, HT-29 KRAS wild-type and HT-29 KRAS mutant cell lines were used. The results of MTT assay indicated that FC induced a stronger dosage-related growth inhibition on HT-29 KRAS mutant than HT-29 KRAS wild-type cells. These data suggest that FC may target one more gene or pathway other than mutant KRAS and thus enhance the cytotoxicity (Synthetic Lethality) of CRC, indicating a critical role of FC on KRAS mutant CRC. In addition, FC induced a higher percentage of cells with acidic vesicular organelles (AVOs, representing an induction of autophagic flux) in HT-29 KRAS mutant cells than HT-29 KRAS wild type cell using flow cytometry. Autophagy inhibitor Bafilomycin A1 (BAF) decreased the percentage of cells with AVOs on both cells. Western blot analysis also showed that FC induced a higher expression of autophagic marker LC-3 II in HT-29 KRAS mutant than HT-29 KRAS wild type cells. Annexin V/propidium iodide double staining analysis showed that both early and late apoptotic cells increased with dosages on both cells, and a stronger apoptosis was triggered on HT-29 KRAS mutant than HT-29 KRAS wild type cells. No induction of necrosis was observed. The immunofluorescence data showed that FC increased the puncta of both Parkin and LC3-II in HT-29 KRAS mutant cells at 48 h of FC treatment. Western blot indicated that no Parkin expression was observed on HT-29 KRAS wild type cells at 48h, suggesting that mitophagy flux maybe completed. Taken together, these data suggest that FC-induced Synthetic Lethality in KRAS mutant cells maybe due to the induction of apoptosis, which maybe reversed by autophagy and mitophagy.