The Mechanism By Which Pien Tze Huang Inhibits Growth Of Colorectal Cancer Stem Cell Via Suppression Of Wnt/β-catenin Pathway

Objective:Detection and isolation the proportion of SP cells in colorectal cancer cell lines SW480, and appraisal biology characteristics of the SP cells to investigate the feasibility of starting research on colorectal cancer stem stem cells (CR-CSCs). To determine the effects of Pien Tze Huang (PZH) on the growth of CR-CSCs and investigate the possible molecular mechanisms.Method:1. Isolation and appraisal of colorectal cancer ctem cells:Cancer stem cells (CSCs) were isolated from SW480 colorectal cancer cell line using SP analysis by FACS with Hoechst 33342 staining. Spheroid formation assay and BALB/c nude mice xenograft experimental were used to examine the SP cells were enriched for CSCs.2. The effect of PZH on the growth of colorectal cancer ctem cells and the possible molecular mechanisms:Phase-contrast microscope was performed to observe the cell morphological changes. MTS assay was evaluated to determine the cell viability of CR-CSCs treatment with PZH. Spheroid formation assay was used to investigate spheroid formation of CR-CSCs. Q-PCR and Western blot were used to determine the mRNA and protein expression of β-catenin and Lgr-5. Immunofluorescence was used to investigate the β-catenin nuclear translocation on CR-CSCs.Result:1. Isolation and appraisal of colorectal cancer ctem cells:SP cells was detected in colorectal cancer cell line SW480 with the proportion of 0.84% ± 0.02%(**P<0.01). Spheroid formation assay showed that the spheroid formation rate of SW480 SP cells were greater than the non-SP cells. BALB/c nude mice xenograft experimental indicated that SP cells shows stronger tumorigenicity than non-SP cells.2. The effect of PZH on the growth of colorectal cancer ctem cells and the possible molecular mechanisms:Phase-contrast microscope results showed that the cell destiny was decreased after treatment with PZH. MTS assay showed that PZH(0.25 mg/mL,0.5 mg/mL, 0.75 mg/mL) respectively reduced the SP cells viability by 30.25%,52.31%,65.90%, compared to untreated cells (*P<0.05). Spheriod formation assay indicated that PZH treatment significantly inhibited the spheriod formation ability of CR-CSCs (*P<0.05). BALB/c nude mice xenograft tumor experimental shows that PZH significantly decrease the tumorigenicity of CR-CSCs. Q-PCR and Western blot analysis showed that PZH significantly down-regulated the expression of β-catenin and Lgr-5 in Wnt/β-catenin signaling pathway (*P<0.05). Immunofluorescence analysis indicated that PZH reduced the β-catenin nuclear translocation.Conclusion:There existing a certain number of SP cells in colorectal cancer cell lines SW480, which can be isolation and used as an research on CR-CSCs. PZH significantly suppressed the growth of CR-CSCs, and decrease activation of Wnt/β-catenin signaling pathway via reducing the β-catenin nuclear translocation and down-regulated the expression of Lgr-5.