Effects Of8-bromo-7-methoxychrysin On Characteristics And Expression Of β-catenin Of Liver Cancer Stem Cells Derived From MHCC97-H Cell Line

Objective:To investigate the effect of8-bromo-7-methoxychrysin(BrMC) on characteristics and expression of β-catenin of liver cancer stem cells derived from MHCC97-H cell line, and explore whether the inhibition of BrMC on the characteristics of LCSCs was dependent on its regulation of the expression of β-catenin.Methods:We sorted CD133+cells from liver cancer MHCC97-H cell line by Magnetic Activated Cell Sorting(MACS), enriched and expanded CD133+sphere forming cells (SFCs) using the stem cell conditioned medium in ultra-low attachment plates. We detected the CD133positive cell percentage by flow cytometry(FCM) with phycoerythrin (PE)-conjugated anti-CD133antibodies. the self-renewal capacity of CD133+SFCs and parental cells was compared by tumorsphere serial passage culture and limited dilution analysis.The expression level of CD133, CD44, E-cadherin, ZO-1, N-cadherin, vimentin, β-catenin in CD133+SFCs and parental cells was analyzed by Western blot. We used the xenograft model in nude mouse in vivo to compare the tumorigenicity of CD133+SFCs and parental cells. LCSCs were treated with various concentrations of BrMC. MTT assay was used to determined the effects of BrMC on the proliferative activity. The inverted microscope was used to observed the effects of BrMC on the single-layer adherent growth morphology of LCSCs. Tumorsphere formation assay was used to evaluate the effects of BrMC on the self-renewal capacity of LCSCs and Matrigel invasion assay in vitro was used to evaluate the effects of BrMC on the invation capability of LCSCs. Western blot was used to analyze the effects of BrMC on the protein expressions of CD133, CD44, E-cadherin, ZO-1, N-cadherin, vimentin and β-catenin. The effects of BrMC targeting for inhibition or eradication of LCSCs in vivo were determined by using primary and secondary xenograft model in Balb/c-nu mice.We examined the effects of BrMC on the self-renewal capability and the expression of β-catenin, CD133and CD44in LCSCs after the treatment of LCSCs with β-catenin specific small interfering RNA(siRNA) or Wnt3a conditioned medium.Results:The results of flow cytometry(FCM) using PE-conjugated anti-CD133antibodies showed that purified CD133+cell was obtained by MACS (CD133+cells:57.29±4.61%; CD133-cells:1.02±0.65%; the parental cells:7.21±1.34%). Tumorspheroids were obtained in both CD133+cells and parental cells, within6days of culture, but the ability of CD133+cells to form tumorspheroids is stronger (number of spheres: CD133+cells:167±31vs parental cells:61±26; volume:CD133+cells:397±45μm3vs parental cells:153±31μm3)(P<0.05), whereas CD133-cells failed to form spheroids under the same condition. The result of tumorsphere serial passage culture showed that SFCs had the ability to serial passage to become tumorspheroids. The result of limited dilution analysis demonstrated that single SFCs could form new tumorspheroids. The result of western blot analysis showed that SFCs over-expressed stem cell markers (CD133and CD44), mesenchymal cell markers (N-cadherin and Vimentin) and β-catenin, however, the expression of epithelial cell markers (E-cadherin and ZO-1)was decreased. We observed that single-layer adherent growth SFCs exhibited a spindle-like shape, while parental cells displayed a cobble-stone-like phenotype under the inverted microscope. The results of Matrigel invasion assay in vitro showed that, compaered with parental cells, SFCs possessed highly invasion ability. The results of the xenograft model in nude mouse in vivo revealed that SFCs possessed high tumorigenicity compared with their parental cells, and the result of HE staining demonstrated that tumor xenografts derived from SFCs had similar histological characteristics with those derived from parental cells, however, the tumor derived from SFCs showed much more CD44and CD133positive cells than that of tumor derived from parental cells. The results of MTT assay demonstrated that BrMC preferentially inhibited proliferation of LCSCs in a dose-dependent manner, with the IC50was0.5μM for SFCs and17.9μM for their parental cells. BrMC(0.1μM) caused morphological changes from mesenchymal cells to epithelial cells in LCSCs. The results of tumorsphere formation assay and secondary passage tumorsphere formation assay showed that BrMC inhibited self-renewal capacity of LCSCs (P<0.05). The reaults of western blot demonstrated that treatment with BrMC (0.1、0.3、1μM) increased the expression of E-cadherin and ZO-1and decreased the he expression of N-cadherin and Vimentin. The results of Matrigel invasion assay in vitro showed that BrMC inhibited the invasiveness capacity of LCSCs in a dose-dependent manner (P<0.05). Western blot analysis showed that BrMC (0.1、0.3、1μM) reduced the expression of CD133and CD44. primary xenograft model in Balb/c-nu mice revealed that BrMC effectively inhibited the growth of the tumors derived from LCSCs (P<0.05) and significantly reduced the CD44and CD133expression frequency in LCSCs-derived tumors. Secondary xenograft model in Balb/c-nu mice showed that cancer cells from vehicle control (refined olive oil)mice exhibited tumor re-growth quickly in secondary mice, while the cancer cells obtained from mice treated with BrMC (50mg/kg) largely failed to re-generrate tumor. Western blot analysis showed that BrMC suppressed the expression of β-catenin in LCSCs. Silencing of β-catenin by siRNA decreased the expression of (3-catenin, CD133and CD44, declined the capacity of tumorspheroids formation (P<0.05) and synergized the expression of β-catenin and the inhibition of tumorsphere formation of LCSCs induced by BrMC(0.1μM)(P<0.05). Pretreatment with Wnt3a conditioned medium increased the expression of β-catenin and stem cell markers(CD133and CD44) and antagonized BrMC-induced inhibition of the expression of β-catenin and the capacity of liver cancer cancer spheroids formation in LCSCs by (P<0.05).Conclusions:1. CD133+SFCs obtained by MACS and stem cell conditioned medium suspension culture possessed the characteristics of liver cancer stem cell.2. BrMC, a novel synthetic analogue of chrysin, has the abilities to inhibit the the fuctions and characteristics of LCSCs.3. BrMC inhibits self-renewal capacity of liver cancer stem cells via down-regulation of β-catenin.