Dianat-Moghadam Hassan, Khalili Mostafa, Keshavarz Mohsen, Azizi Mehdi, Hamishehkar Hamed, Rahbarghazi Reza, Nouri Mohammad
Department of Medical Biotechnology, Faculty of Advanced Medical Sciences, Tabriz University of Medical Sciences, Tabriz, Iran.
Stem Cell Research Center, Tabriz University of Medical Sciences, Tabriz, Iran.
Cancer Cell Int. 2021 Feb 10;21(1):100. doi: 10.1186/s12935-021-01803-4.
The expansion and metastasis of colorectal cancers are closely associated with the dynamic growth of cancer stem cells (CSCs). This study aimed to explore the possible effect of LXR (a regulator of glycolysis and lipid hemostasis) in the tumorgenicity of human colorectal CD133 cells.
Human HT-29 CD133 cells were enriched by MACS and incubated with LXR agonist (T0901317) and antagonist (SR9243) for 72 h. Cell survival was evaluated using MTT assay and flow cytometric analysis of Annexin-V. The proliferation rate was measured by monitoring Ki-67 positive cells using IF imaging. The modulation of LXR was studied by monitoring the activity of all factors related to ABC transporters using real-time PCR assay and western blotting. Protein levels of metabolic enzymes such as PFKFB3, GSK3β, FASN, and SCD were also investigated upon treatment of CSCs with LXR modulators. The migration of CSCs was monitored after being exposed to LXR agonist using scratch and Transwell insert assays. The efflux capacity was measured using hypo-osmotic conditions. The intracellular content of reactive oxygen species was studied by DCFH-DA staining.
Data showed incubation of CSCs with T0901317 and SR9243 reduced the viability of CD133 cells in a dose-dependent manner compared to the control group. The activation of LXR up-regulated the expression and protein levels of ABC transporters (ABCA1, ABCG5, and ABCG8) compared to the non-treated cells (p < 0.05). Despite these effects, LXR activation suppressed the proliferation, clonogenicity, and migration of CD133 cells, and increased hypo-osmotic fragility (p < 0.05). We also showed that SR9243 inhibited the proliferation and clonogenicity of CD133 cells through down-regulating metabolic enzymes PFKFB3, GSK3β, FASN, and SCD as compared with the control cells (p < 0.05). Intracellular ROS levels were increased after the inhibition of LXR by SR9243 (p < 0.05). Calling attention, both T0901317 and SR9243 compounds induced apoptotic changes in cancer stem cells (p < 0.05).
The regulation of LXR activity can be considered as a selective targeting of survival, metabolism, and migration in CSCs to control the tumorigenesis and metastasis in patients with advanced colorectal cancers.
结直肠癌的扩张和转移与癌干细胞（CSCs）的动态生长密切相关。本研究旨在探讨肝脏X受体（LXR，糖酵解和脂质稳态的调节因子）对人结直肠CD133细胞致瘤性的可能影响。
通过磁珠分选法富集人HT-29 CD133细胞，并用LXR激动剂（T0901317）和拮抗剂（SR9243）孵育72小时。使用MTT法和膜联蛋白V的流式细胞术分析评估细胞存活率。通过免疫荧光成像监测Ki-67阳性细胞来测量增殖率。使用实时PCR测定法和蛋白质印迹法监测与ABC转运蛋白相关的所有因子的活性，研究LXR的调节作用。在用LXR调节剂处理CSCs后，还研究了代谢酶如磷酸果糖激酶-2/果糖-2,6-二磷酸酶3（PFKFB3）、糖原合成酶激酶3β（GSK3β）、脂肪酸合酶（FASN）和硬脂酰辅酶A去饱和酶（SCD）的蛋白质水平。使用划痕试验和Transwell小室试验监测CSCs在暴露于LXR激动剂后的迁移情况。使用低渗条件测量外排能力。通过二氯荧光素二乙酸酯（DCFH-DA）染色研究细胞内活性氧的含量。
数据显示，与对照组相比，用T0901317和SR9243孵育CSCs以剂量依赖性方式降低了CD133细胞的活力。与未处理的细胞相比，LXR的激活上调了ABC转运蛋白（ABCA1、ABCG5和ABCG8）的表达和蛋白质水平（p < 0.05）。尽管有这些作用，但LXR激活抑制了CD133细胞的增殖、克隆形成能力和迁移，并增加了低渗脆性（p < 0.05）。我们还表明，与对照细胞相比，SR9243通过下调代谢酶PFKFB3、GSK3β、FASN和SCD来抑制CD133细胞的增殖和克隆形成能力（p < 0.05）。SR9243抑制LXR后细胞内活性氧水平升高（p < 0.05）。值得注意的是，T0901317和SR9243两种化合物均诱导癌干细胞发生凋亡变化（p < 0.05）。
LXR活性的调节可被视为对CSCs的存活、代谢和迁移进行选择性靶向，以控制晚期结直肠癌患者的肿瘤发生和转移。
