| Cancer has been becoming the most serious public problem in worldwide, where lung cancer, gastric cancer and liver cancer are top three causes to cancer-relate mortality. Due to high motility of liver cancer, its morbidity is steadily rising, thus treatments, prevention and the related mechanism exploration of cancers have been being a significant scientific issue for tumor research. The main treatments for primary liver cancer including hepatic resection, radiofrequency ablation, chemotherapy and liver transplantation, but all those means are not sufficient to effectively inhibit recurrence and metastasis, making treatments of liver cancer still a greater challenge.Cancer stem cells(CSCs), which are a small subpopulation of cells in tumors with proliferation capability and stem-like properties and differentiation, have great potential of self-renewal and differentiation. LCSCs are essential for tumor initiation, and play crucial roles in recurrence, metastasis and poor prognosis of cancer. Therapeutic applications targeting LCSCs would be a brand new strategy for curing cancer.Cancer initiation and progression are a complex process controlled by multiple factors, several force and chemical factors are involved in. inhibition of CSCs metastasis and promotion of differentiation are important means to target CSCs in cancer therapy, while little is known about the effects of mechanical and chemical factors on biological behaviors like metastasis and differentiation of LCSCs. In this article, biomechanical properties of LCSCs are first assessed, following research focus on the influence of mechanical factors(shear stress, SS) and chemical factors(salinomycin, Sal) on metastasis and differentiation of LCSCs and molecular basis involved in. Main contents and results are described as follow:(1) Enrichment, identification and biomechanical profile of LCSCsLCSCs are purified with cultivation on serum free stem cell medium from MHCC97 H cells. Obtained cells are subject to flowcytometry and drug sensitive test, clone formation assay, tumorigenicity experiments to identify if they are LCSCs and find that CSC surface markers CD133, CD90 and Oct3/4 are expressed significantly higher than their MHCC97 H counterparts. Laser scanning confocal microscope demonstrate a punctiform F-actin distribution of LCSCs in comparation of filiform structure of MHCC97 H, besides, F-actin is found to be less in LCSCs than in MHCC97 H by applying western bolt. Theseresults strongly suggesting a close relevanceof liver cancer biomechanical properties and the metastasis capability.(2) Shear stress promotes LCSCs migration via FAK-ERK-β-catenin signaling pathwayShear stress with 2 dyne/cm2, which was produced by the flow medium, was exerted on LCSCs. After 6 h, 2 dyne/cm2 shear stress loading, transwell essay and western blot separately demonstrated that LCSCs migration capability is promoted, accompany with up-regulated level of β-catenin and phosphorylated FAK and ERK1/2, but shear stress promoted cell migration is suppressed when FAK and ERK1/2 activation and β-catenin expression are interference. By using immunofluorescence, rearranged F-actin fiber is observed in cells subjected to shear stress but inhibition of FAK, ERK1/2 or β-catenin up-regulated F-actin expression. AFM demonstrated that the shear stress treatment loading significantly reduced young’s modulus of LCSCS, but inhibition of FAK, ERK1/2 and β-catenin respectively elevated the shear stress reduced cell stiffness. Furthermore, FAK inhibitor in this experiment could affect ERK phosphorylation and β-catenin expression, suggesting that LCSCs is regulated by shear stress via FAK-ERK1/2-β-catenin pathway. Converging, these results suggest that shear stress promotes LCSCs migration via rearranging cytoskeleton by activating FAK/ERK1/2/β-catenin signal pathway.(3) Shear stress regulates LCSCs differentiation via Wnt/β-catenin signaling pathwayFlowcytometry are applied to detect the expression of LCSCs surface markers CD133, CD90 and Oct3/4 after LCSCs are subject to 2 dyne/cm2 shear stress for 2days,results indicate that CD133, CD90 and Oct3/4 decreased after the shear stress treatment. Self-renewing capability is assessed with drug sensitivity essay and find that shear stress treated LCSCs are more sensitive to cisplatin and 5-FU with less survival rate. Moreover, AFM experiment demonstrated an increased young’smodulus of shear stress treated LCSCs.Western blot suggests thatβ-catenin expression in the shear stress treated LCSCs significantly decreased, but CD133, CD90 and Oct3/4 recover to a extend after artificial activation ofβ-catenin.These results indicate that 2days loading of 2 dyne/cm2 shearstress could down-regulate Wnt/β-cateninpathway in LCSCs to decrease the cancer stem cell markers CD133, CD90 and Oct3/4 expression,decrease the sphere-forming capability,increase drug sensitivity and modulate cell stiffness, suggesting that LCSCs differentiated after the shear stress treatment.(4) Salinomycin regulates motility of LCSCs via FAF-ERK1/2 signaling pathwayTranswell assay demonstrates that Sal decreased LCSCs migration and invasion in given concentration with a concentration dependent manner. FAK and ERK1/2 phosphorylation are found to be down-regulated by western blot after LCSCs are treated with Sal. Further study show that FAK, ERK1/2 inhibitors suppressed LCSCs migration and invasion as expected, suggesting Sal suppresses LCSCs migration and invasion via FAK-ERK1/2 pathway. MMP-2 and MMP-9 activity are assessed with gelatin zymology and find that Sal suppressed the secretion of MMP-2 and MMP-9.In terms of the mechanical properties, Young’s modulus of LCSCs was decreased by treatment of Sal, and obvious formation of F-actinfiliform structure was also observed in Sal-treated LCSCs by Immunofluoresence staining. Inhibitor of FAK or ERK1/2 could also decrease of Young’s modulus and increase the formation of F-actinfiliform structure. Overall, our study demonstrated that Sal inhibitsLCSCs motility by lessening cell stiffness, increasing F-actin expression and MMP-2, MMP-9 secretion via the FAK-ERK1/2 signaling pathway.(5) Salinomycin modulates LCSCsdifferentiation via Wnt/β-catenin signaling pathwayFlowcytometry are applied to examine cancer stem cell surface marker CD133, CD90 and Oct3/4 expression of LCSCs subjected to 2 days of Sal treatment.Results showed that Sal treatment significantly decreased CD133, CD90 and Oct3/4 expression. Self-renewing capability of Sal treated LCSCs is assessed with sphere-forming experiment, results demonstrated that 2 days of Sal treatment significantly suppressed the sphere forming capability, but which is, as expected, recovered to an extend after Wnt/β-catenin is activated with LiCl. Sal treated LCSCs are found to be more sensitive to cisplatin and 5-FU and with less survival rate by applying drug sensitivity experiment. AFM experiment indicate that Sal treated LCSCs notably increased young’s modulus, which is, together with cancer stem cell marker CD133, CD90 and Oct3/4, recovered after artificial activation of β-catenin. A tumorigenicity experiment reveals that tumor size significantly diminished after the Sal treatment. These results indicate that the Sal treatment could down-regulate Wnt/β-catenin pathway to decrease CD133, CD90 and Oct3/4 expression and sphere-forming ability, subsequently increase the drug sensitivity and modulate cell stiffness. All evidences are strongly suggesting the differentiation of LCSCs after Sal treatment.To conclude, a shear stress and drug stimulus plays pivotal roles in modulation of LCSCs metastasis and differentiation. Our research offer not only experimental basis of in-sight in the effects of mechanical and chemical factors on LCSCs and the related mechanism, but also theoretical cues for LCSCs targeted clinical applications. |
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