Figure 1.
The effect of DLF on HCC cell growth and cell cycle-related protein expression in vitro.
(A, B) Growth inhibition resulting from the treatment of HCC cells with DLF extracts for 24 h and 48 h. (C) The cell cycle distribution of Huh7 and MHCC-97L cells that were treated with 3 mg/ml DLF for 24 h or 48 h. (D) The percentage of Huh7 cells in G2 phase following treatment with DLF extracts for various times. (E, F) Western blot analysis of the expression of G2/M phase transition-related proteins after treatment with DLF extracts at various concentrations and for various times.
Figure 2.
DLF extracts causes cell growth inhibition and apoptosis.
(A) The growth inhibition rates of Huh7, SMMC-7721, PLC/PFR/5 and L-02 cells resulting from treatment with ICD for 48 h. (B) Following the treatment of Huh7 cells with 0, 100, 200 or 300 µg/ml ICD for 48 h, apoptotic cells were detected by Annexin V and 7-AAD double staining. (C) Western blot analysis of cleaved PARP in Huh7, SMMC-7721 and PLC/PRF/5 cells following ICD treatment at their respective IC50 values (250 µg/ml, 200 µg/ml, 250 µg/ml). (D) An amount of 1×106 Huh7 or SMMC-7721 cells/mouse was subcutaneously injected into nude mice. After 2 weeks, 0.4 mg/ml of ICD or PBS was administered 5 times per week for 4 weeks. The resulting tumors were excised from the animals after treatment. (E) The tumor weights for the four groups of animals were compared, and statistical significance was determined using the Student’s t-test. Each point represents the mean ± SD.
Figure 3.
ICD treatment causes cells cycle arrest at the G2/M phase.
(A) The cell cycle distribution of Huh7 cells that were treated with various doses of ICD for 18 h. (B) A statistical graph of the cell cycle distribution shown in (A). (C and D) Western blot analysis of G2/M transition-related proteins after ICD treatment of Huh7, SMMC-7721 and PLC/PRF/5 cells for 18 h.
Table 1.
Cell cycle distribution of Huh7 HCC cells after ICD treatment with gradient concentrations.
Table 2.
Cell cycle distribution of SMMC-7721 HCC cells after ICD treatment with gradient concentrations.
Figure 4.
The relative contributions of the Chk1, Chk2 and p53 pathways.
(A) Western blot analysis of the activation of p53, MDM2, Chk1 and Chk2 in Huh7 and PLC/PFR/5 cells after ICD treatment for 48 h. (B) The cell cycle distribution of Huh7 cells after ICD treatment for 18 h following siRNA transfection. (C) Western blot analysis of the silencing effect of Chk1 or Chk2 expression levels after transfection with their corresponding siRNA. (D) Western blot analysis of p-CDK1 or CDK1 expression levels after ICD treatment for 18 h following transfection with their corresponding siRNA.
Figure 5.
The effect of growth suppression on CD133+/− PLC/PRF/5.
(A) PLC/PRF/5 cells were treated with various doses of ICD or 10 ng/ml vincristine for 48 h. The percentage of CD133+ cells was then determined by flow cytometry. Statistically significant differences were determined using the Student’s t-test (* = p<0.05; each point represents the mean ± SD). (B) The hepatosphere formation of PLC/PFR/5 CD133+/− cells that were sorted by FACS and treated with PBS or 150 µg/ml of ICD for 48 h. (C) The colony formation of PLC/PRF/5 CD133+/− cells that were sorted by FACS and then treated with PBS or 150 g/ml ICD for 48 h. (D) The growth inhibition rates of PLC/PFR/5 CD133+/− cells resulting from 48 and 72 h ICD treatments. (E and F) An amount of 5000 PLC/PFR/5 CD133+/− cells/mouse were subcutaneously injected into nude mice. After 2 weeks, 0.4 mg/ml of ICD or PBS was administered 5 times per week for 4 weeks. The resulting tumors were excised from the animals after treatment. Statistical significance was determined using the Student’s t-test. Each point represents the mean ± SD.