Background Vasculogenic mimicry (VM) is characterized by the formation of tubular structure inside the tumor stroma. It has been shown that a small fraction of cancer cells, namely cancer stem cells (CSCs), could stimulate the development of vascular units in the tumor niche, leading to enhanced metastasis to the remote sites. This study aimed to study the inhibitory effect of phytocompound, Thymoquinone (TQ), on human breast MDA-MB-231 cell line via monitoring Wnt/PI3K signaling pathway. Methods MDA-MB-231 CSCs were incubated with different concentrations of TQ for 48 h. The viability of CSCs was determined using the MTT assay. The combination of TQ and PI3K and Wnt3a inhibitors was examined in CSCs. By using the Matrigel assay, we measured the tubulogenesis capacity. The percent of CD24− CSCs and Rhodamine 123 efflux capacity was studied using flow cytometry analysis. Protein levels of Akt, p-Akt, Wnt3a, vascular endothelial-cadherin (VE-cadherin), and matrix metalloproteinases-2 and -9 (MMP-2 and -9) were detected by western blotting. Results TQ decreased the viability of CSCs in a dose-dependent manner. The combination of TQ with PI3K and Wnt3a inhibitors reduced significantly the survival rate compared to the control group (p < 0.05). TQ could blunt the stimulatory effect of vascular endothelial growth factor (VEGF), epidermal growth factor (EGF), fibroblast growth factor (FGF) on CSCs (p < 0.05). The vasculogenic capacity of CSCs was reduced after being-exposed to TQ (p < 0.05). Western blotting revealed the decrease of CSCs metastasis by suppressing MMP-2 and -9. The protein level of VE-cadherin was also diminished in TQ-treated CSCs as compared to the control cell (p < 0.05), indicating inhibition of mesenchymal-endothelial transition (MendT). TQ could suppress Wnt3a and PI3K, which coincided with the reduction of the p-Akt/Akt ratio. TQ had the potential to decrease the number of CD24− CSCs and Rhodamine 123 efflux capacity after 48 h. Conclusion TQ could alter the vasculogenic capacity and mesenchymal-epithelial transition of human breast CSCs in vitro. Thus TQ together with anti-angiogenic therapies may be a novel therapeutic agent in the suppression of VM in breast cancer.
Background Here, we examined the tumoricidal effect of Gemini surfactant nanoparticles enriched with curcumin on 3D spheroid HT-29 cells. The delivery of curcumin and other phytocompounds to the tumor niche is an important challenge. Methods Spheroid HT-29 cells were generated by using a conventional hanging drop method and exposed to different concentrations of Gemini-curcumin nanoparticles. The changes in spheroid integrity and cell viability were evaluated by measuring the spheroid diameter and LDH release, respectively. The uptake of Gemini-curcumin nanoparticles was detected by flow cytometry assay. Flow cytometric of Rhodamine 123 efflux was also performed. Migration capacity was analyzed using a Transwell insert assay. By using real-time PCR analysis and Western blotting, we studied the expression level of MMP-2, -9, Vimentin, and E-cadherin genes. Results Gemini-curcumin nanoparticles had the potential to disintegrate spheroids and decrease central density compared to the control group (p < 0.05). These changes coincided with enhanced LDH release by the increase of nanoparticle concentration (p < 0.05). Data highlighted the ability of cells to uptake synthetic nanoparticles in a dose-dependent manner. We found reduced Rhodamine 123 efflux in treated HT-29 spheroid cells compared to the control (p < 0.05). Nanoparticles significantly decreased the metastasis and epithelial-mesenchymal transition (EMT) rate by the suppression of MMP-2 and MMP-9, Vimentin, and induction of E-cadherin (p < 0.05). Conclusion Our data confirmed that Gemini curcumin has the potential to suppress cell proliferation and inhibit metastasis in 3D spheroid HT-29 cells in vitro.
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