Abstract
To investigate the effect and mechanism of SB525334 on self-renewal, migration and invasion of ovarian cancer stem cells. ALDHhigh-expressing cancer stem cells (CSCs) were isolated from human ovarian cancer cell line SKOV-3 by flow cytometry and treated with 2μg/mL SB525334 for 6h. The sphere forming assay was used to detect the ability of self-renewal of CSCs and the colony formation assay was used to detect the tumorigenicity in vitro. Transwell migration and invasion assay were used to detect the migration and invasion ability of CSCs. To further explore the mechanism, real-time quantitative PCR and flow cytometry were used to detect the mRNA and protein expression of TGF-β, Smad2, Smad3, phosphorylated Smad2, phosphorylated Smad3 and Smad4, respectively. Expressions of epithelial-mesenchymal transition (EMT)-related genes E-cadherin, Snail, Vimentin were also assessed. The self-renewal ability, tumorigenicity in vitro, migration and invasion ability of CSCs were significantly attenuated after SB525334 treatment. The expressions of TGF-β, phosphorylated Smad2, phosphorylated Smad3, Snail, and Vimentin were decreased, while Smad4 and E-cadherin expressions were increased. SB525334 may inhibit the self-renewal, invasion and migration of ovarian CSCs by blocking the TGF-β/Smad/EMT pathway.
Highlights
The self-renewal ability, tumorigenicity in vitro, migration and invasion ability of cancer stem cells (CSCs) were significantly attenuated after SB525334 treatment
We first sorted out ALDHhigh and ALDHlow cells (Fig 1A), and used sphere forming assay and plate colony formation assay to identify tumor stem cell characteristics
The results show that ALDHhigh and ALDHlow cells have the spheroid numbers of 345±19.52 and 57.67 ±7.09 (t = 23.96, P = 0.0001) (Fig 1B); the numbers of colony formation are 46±4.58 and 26.67 ±2.08 (t = 6.65, P = 0.0027), respectively (Fig 1C)
Summary
The sorted ALDHhigh CSCs were cultured in serum-free DMEM/F12 medium containing 5 mg/mL insulin, 0.4% BSA, 2% B27, 10 ng/mL FGF, and 10 ng/mL EGF for 48 h and subjected to subsequent experiments. 200 cancer stem cells were inoculated into 6-well plates of McCoy’s 5a medium containing 10% fetal bovine serum for 7 days, rinsed twice with PBS, fixed in 75% ethanol for 15 min at room temperature, stained with crystal violet at room temperature for 15 min, rinsed 4 times with PBS. They were photographed and counted for colony formation for statistical analysis. The average fluorescence intensity ratio is proportional to the amount of protein expression, which can be obtained from the ratio of mean fluorescence intensity of the samples to the corresponding average fluorescence intensity of the isotype control samples
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
