Lei Nan, Xiong Si-Hui, Tan Li, He Man, Zhang Meng, Sun Qiang, Zeng Sha, Chen Li, Zhou Li-Juan, Xu Hai-Bo
Department of Pharmacology, School of Pharmacy, Chengdu University of Traditional Chinese Medicine Chengdu 611137, China.
Sichuan Academy of Chinese Medical Sciences Chengdu 610041, China.
Zhongguo Zhong Yao Za Zhi. 2020 Apr;45(7):1676-1683. doi: 10.19540/j.cnki.cjcmm.20200108.401.
The objective of this study was to investigate the inhibitory effect of scutellarin on the differentiation of colonic cancer stem cells in vitro and in vivo and to explore its underlying hedgehog signaling-based mechanism. The effect of scutellarin on the growth in vitro of HT-29 cells-derived cancer stem-like cells(HT-29 CSC) was observed with 3 D cell culture. The effect of scutellarin on the transformation of HT-29 CSC cells was assessed by soft agar colony formation assay. Fetal calf serum was used to induce differentiation of stem cells and observe the effect of scutellarin on HT-29 CSC cells differentiation in vitro. The effects of scutellarin on mRNA expressions of Lgr5, c-Myc, CK20 and Nanog in HT-29 CSC cells were determined by quantitative Real-time polymerase chain reaction(qRT-PCR). The effects of scutellarin on protein expressions of c-Myc, Gli1 and Lgr5 in HT-29 CSC cells were examined by Western blot. After subcutaneous implantation of HT-29 CSC cells in nude mice, the effect of scutellarin on the mouse body weight and the growth of HT-29 CSC-derived tumor were explored. qRT-PCR was used for evaluating the effect of scutellarin on mRNA levels of CD133, Lgr5, Gli1, Ptch1, c-Myc, Ki-67, CK20 and Nanog in tumor. Western blot and immunohistochemistry analysis were used to detect the effect of scutellarin on protein expressions of c-Myc, Gli1, Lgr5, CD133 and Ki-67 in tumor. The in vitro experiments showed that scutellarin inhibited the growth, transformation and differentiation of HT-29 CSC cells, significantly down-regulated the mRNA levels of Lgr5, c-Myc, CK20 and Nanog in HT-29 CSC cells as well as the protein expression levels of c-Myc, Gli1 and Lgr5 in HT-29 CSC cells. Additionally, animal experiments showed that scutellarin significantly inhibited the growth of subcutaneous xenografts in nude mice, and down-regulated the mRNA expressions of CD133, Lgr5, Gli1, Ptch1, c-Myc, Ki-67, CK20 and Nanog as well as the protein levels of c-Myc, Gli1, Lgr5, CD133 and Ki-67 of xenografts in nude mice. Taken together, scutellarin could inhibit the differentiation of colo-nic cancer stem cells in vitro and in vivo, potentially by down regulation of hedgehog signaling pathway activity.
本研究的目的是探讨灯盏花素在体外和体内对结肠癌干细胞分化的抑制作用，并探索其基于刺猬信号通路的潜在机制。采用三维细胞培养观察灯盏花素对HT-29细胞来源的癌干细胞样细胞（HT-29 CSC）体外生长的影响。通过软琼脂集落形成试验评估灯盏花素对HT-29 CSC细胞转化的影响。用胎牛血清诱导干细胞分化，观察灯盏花素对HT-29 CSC细胞体外分化的影响。通过定量实时聚合酶链反应（qRT-PCR）测定灯盏花素对HT-29 CSC细胞中Lgr5、c-Myc、CK20和Nanog mRNA表达的影响。通过蛋白质印迹法检测灯盏花素对HT-29 CSC细胞中c-Myc、Gli1和Lgr5蛋白表达的影响。将HT-29 CSC细胞皮下接种于裸鼠后，探讨灯盏花素对小鼠体重及HT-29 CSC来源肿瘤生长的影响。用qRT-PCR评估灯盏花素对肿瘤中CD133、Lgr5、Gli1、Ptch1、c-Myc、Ki-67、CK20和Nanog mRNA水平的影响。采用蛋白质印迹法和免疫组织化学分析检测灯盏花素对肿瘤中c-Myc、Gli1、Lgr5、CD133和Ki-67蛋白表达的影响。体外实验表明，灯盏花素可抑制HT-29 CSC细胞的生长、转化和分化，显著下调HT-29 CSC细胞中Lgr5、c-Myc、CK20和Nanog的mRNA水平以及HT-29 CSC细胞中c-Myc、Gli1和Lgr5的蛋白表达水平。此外，动物实验表明，灯盏花素可显著抑制裸鼠皮下异种移植瘤的生长，并下调裸鼠异种移植瘤中CD133、Lgr5、Gli1、Ptch1、c-Myc、Ki-67、CK20和Nanog的mRNA表达以及c-Myc、Gli1、Lgr5、CD133和Ki-67的蛋白水平。综上所述，灯盏花素可能通过下调刺猬信号通路活性在体外和体内抑制结肠癌干细胞的分化。
