Role of ursolic acid chalcone, a synthetic analogue of ursolic acid, in inhibiting the properties of CD133(+) sphere-forming cells in liver stem cells

Abstract

The expression of CD133 decreases with differentiation of tumor cell, indicating that CD133 is a specific marker for isolation and identification of CSCs. In the present study the effect of Ursolic acid chalcone (UAC) on CD133(+) hepatocellular carcinoma cell (HCC CSCs) differentiation, their self-renewal, tumorigenic capacity and sensitivity to chemotherapeutic drugs was studied. The results demonstrated that UAC inhibits the expression of CD133(+) in a dose and time-dependent manner in PLC/PRF/5 and Huh7 HCC cells. The inhibition was significant at 50 μM and on day 8. The percentage of CD133(+) cells decreased from an initial 59.3% in PLC/PRF/5 to 37.1% and 78.2% in Huh7 to 59.2% on treatment with UAC. There was inhibition of Oct4, Tert, Bmi1, β-catenin, ABCG2, and tumor sphere-related gene Ep300. In addition it also decreased number of CK19-positive cells and increased number of CK8/18-positive cells. UAC treatment caused a decrease in self-renewal capability and increase in sensitivity to doxorubicin and vincristine drugs in CD133(+) HCC CSCs. Therefore, UAC can be a potent therapeutic agent to target differentiation of CSC in HCC.

Keywords: Self-renewal; cell differentiation; hepatocellular carcinoma; therapeutic agent.

Figure 1

Structure of Ursolic acid chalcone.

Figure 2

Decrease in CD133⁺ CSCs population by UAC in vitro. A. Western blotting for CD133 in PLC/PRF/5 cells on treatment with different doses of UAC. B. UAC induces decrease in CD133 protein in a time-dependent manner. C. Effect of different concentrations of UAC on PLC/PRF/5 cell proliferation (BrdUrd ELISA assay). D. Reduction in percentage of CD133⁺ cells after UAC treatment (Flow cytometric analysis).

Figure 3

Induction of differentiation in HCC CSCs by UAC. A. The stem-ness-related gene expression of in CD133⁺ and CD133^- cells, cultured as spheres in CDM or in a monolayer with UAC treatment. B. Western blotting shows that UAC enhances CK8/18 expression and decreases CK19 expression in a time dependent manner.

Figure 4

Inhibition of self-renewal and tumorigenic capacities of CD133⁺ CSCs by UAC. A. CD133⁺ and CD133^- PLC/PRF/5 cells were pretreated with UAC for 10 days. Each group of cells was suspended in growth media containing 0.3% soft agar and seeded in 24-well plates to evaluate colony formation efficiency (CFE; n = 3). B. Inhibition of capacity for CD133⁺ PLC/PRF/5 cell sphere formation by UAC.

Figure 5

Enhancement in the activity of chemotherapeutic agents on HCC CSCs by UAC. The cytotoxic effects of doxorubicin and vincristine on CD133⁺ and CD133^- PLC/PRF/5 cells that had been pretreated with UAC were tested with the MTT assay.