Wei Fang, Zhang Tong, Yang Zhi, Wei Jian-Chang, Shen Hong-Fen, Xiao Dong, Wang Qiang, Yang Ping, Chen Hua-Cui, Hu He, Chen Zhuan-Peng, Huang Qing, Li Wang-Lin, Cao Jie
Guangzhou Digestive Disease Center, Guangzhou First People's Hospital, Guangzhou Medical University, Guangzhou, China.
Guangzhou Digestive Disease Center, Guangzhou First People's Hospital, the Second Affiliated Hospital, South China University of Technology, Guangzhou, China.
Cell Physiol Biochem. 2018;46(2):829-846. doi: 10.1159/000488740. Epub 2018 Mar 29.
BACKGROUND/AIMS: Gambogic acid (GA), the main active compound of Gamboge hanburyi, has been reported to be a potential novel antitumor drug. Whether GA inhibits putative cancer stem cells (CSCs), which are considered to be the major cause of cancer treatment failure, remains largely unknown. This study investigated whether GA inhibits the CSCs of colorectal cancer (CRC) and its possible mechanisms.
We performed CCK8 and tumor sphere formation assays, percentage analysis of both side population and CD133+CD44+ cells, and the detection of stem cells markers, in order to assess the role of GA in inhibiting the stem celllike features of CRC. An mRNA microarray was performed to identify the downstream gene affected by GA and rescue assays were performed to further clarify whether the downstream gene is involved in the GA induced decrease of the stem cell-like CRC population. CRC cells were engineered with a CSC detector vector encoding GFP and luciferase (Luc) under the control of the Nanog promoter, which were utilized to investigate the effect of GA on putative CSC in human tumor xenograft-bearing mice using in vivo bioluminescence imaging.
Our results showed that GA significantly reduced tumor sphere formation and the percentages of side population and CD133+CD44+ cells, while also decreasing the expression of stemness and EMT-associated markers in CRC cells in vitro. GA killed stem-like CRC cells by upregulating the expression of ZFP36, which is dependent on the inactivation of the EGFR/ ERK signaling pathway. GFP+ cells harboring the PNanog-GFP-T2A-Luc transgene exhibited CSC characteristics. The in vivo results showed that GA significantly inhibited tumor growth in nude mice, accompanied by a remarkable reduction in the putative CSC number, based on whole-body bioluminescence imaging.
These findings suggest that GA significantly inhibits putative CSCs of CRC both in vitro and in vivo by inhibiting the activation of the EGFR/ ERK/ZFP36 signaling pathway and may be an effective drug candidate for anticancer therapies.
背景/目的：藤黄酸（GA）是藤黄的主要活性成分，据报道是一种潜在的新型抗肿瘤药物。GA是否能抑制被认为是癌症治疗失败主要原因的假定癌症干细胞（CSCs），在很大程度上仍不清楚。本研究调查了GA是否能抑制结直肠癌（CRC）的CSCs及其可能的机制。
我们进行了CCK8和肿瘤球形成试验、侧群细胞和CD133+CD44+细胞的百分比分析以及干细胞标志物的检测，以评估GA在抑制CRC干细胞样特征中的作用。进行mRNA微阵列以鉴定受GA影响的下游基因，并进行拯救试验以进一步阐明下游基因是否参与GA诱导的CRC干细胞样群体减少。用在Nanog启动子控制下编码绿色荧光蛋白（GFP）和荧光素酶（Luc）的CSC检测载体对CRC细胞进行工程改造，利用体内生物发光成像研究GA对人肿瘤异种移植小鼠中假定CSC的影响。
我们的结果表明，GA显著减少肿瘤球形成以及侧群细胞和CD133+CD44+细胞的百分比，同时还降低了体外CRC细胞中干性和上皮-间质转化相关标志物的表达。GA通过上调ZFP36的表达杀死干细胞样CRC细胞，这依赖于表皮生长因子受体（EGFR）/细胞外信号调节激酶（ERK）信号通路的失活。携带PNanog-GFP-T2A-Luc转基因的GFP+细胞表现出CSC特征。体内结果表明，基于全身生物发光成像，GA显著抑制裸鼠肿瘤生长，同时假定CSC数量显著减少。
这些发现表明，GA通过抑制EGFR/ERK/ZFP36信号通路的激活，在体外和体内均显著抑制CRC的假定CSCs，可能是一种有效的抗癌治疗候选药物。
