Reza Rahbarghazi1,2,3, Mohammad Nouri4,5,6, Sanya Haiaty7,8, Mohammad-Reza Rashidi9, Maryam Akbarzadeh7,10, Ahad Bazmany11,12, Mostafa Mostafazadeh7,8, Saba Nikanfar7,8, Zohre Zibaei8,12. 1. Stem Cell and Regenerative Medicine Institute, Tabriz University of Medical Sciences, Tabriz, Iran. Rezarahbardvm@gmail.com. 2. Stem Cell Research Center, Tabriz University of Medical Sciences, Imam Reza St., Golgasht St, Tabriz, Iran. Rezarahbardvm@gmail.com. 3. Departmnt of Applied Cell Sciences, Faculty of Advanced Medical Sciences, Tabriz University of Medical Sciences, Tabriz, Iran. Rezarahbardvm@gmail.com. 4. Department of Biochemistry and Clinical Laboratories, Tabriz University of Medical Sciences, Tabriz, Iran. Nourimd@yahoo.com. 5. Stem Cell and Regenerative Medicine Institute, Tabriz University of Medical Sciences, Tabriz, Iran. Nourimd@yahoo.com. 6. Stem Cell Research Center, Tabriz University of Medical Sciences, Imam Reza St., Golgasht St, Tabriz, Iran. Nourimd@yahoo.com. 7. Department of Biochemistry and Clinical Laboratories, Tabriz University of Medical Sciences, Tabriz, Iran. 8. Student Research Committee, Tabriz University of Medical Sciences, Tabriz, Iran. 9. Stem Cell and Regenerative Medicine Institute, Tabriz University of Medical Sciences, Tabriz, Iran. 10. Department of Biochemistry, Erasmus University Medical Center, Rotterdam, the Netherlands. 11. Department of Pathobiology, Faculty of Veterinary Medicine, Ferdowsi University Of Mashhad, Mashhad, Iran. 12. Research Center of Infectious Diseases and Tropical Medicine, Tabriz University of Medical Science, Tabriz, Iran.
Abstract
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: 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 humanbreast CSCs in vitro. Thus TQ together with anti-angiogenic therapies may be a novel therapeutic agent in the suppression of VM in breast cancer.
Entities:
Keywords:
Breast Cancer stem cells; Thymoquinone; Vasculogenic mimicry; Wnt3a/PI3K signaling pathways
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