Fig 1.
DHM combination with NDP inhibits the ability of colony formation of liver cancer cells.
(A) Colony formation capability assay with different treatments of DHM and NDP in HCC cells. The images were captured with a Nikon camera (Japan). (B) The clones were quantified and presented as a statistical figure. (C, D) The proliferation-toxicity of NDP or DHM in QGY7701, HL7702 and SMMC7721 cells with various drug concentrations and treatment durations were assayed using the MTT method (means ± S.D). (D) Proliferation and toxicity of different drug combinations were measured using the MTT assay (means ± S.D). Each experiment was repeated at least three times.
Fig 2.
Synergistic effect of DHM and NDP promotes liver cancer cell apoptosis.
(A) The effect of each drug individually and in combination on cell apoptosis in QGY7701, HL7702 and SMMC7721 cells with different concentrations and treatment durations were monitored under microscopy (100×). (B, C) The apoptosis of QGY7701, HL7702 and SMMC7721 cells induced by the drugs individually and in combination at different concentrations and treatment durations were detected using FITC Annexin V-PI Apoptosis detection kit (BD Pharmingen, USA) and measured by flow cytometry analysis (means ± S.D). Each experiment was repeated at least three times.
Fig 3.
Apoptotic signaling pathway protein expression was evaluated using western blotting.
(A) Apoptotic-related protein expression after drug exposure in QGY7701, HL7702 and SMMC7721 cells was quantitated by western blot. (B, C) The Bcl-2/Bax and Bcl-2/Bak protein ratios were measured by optical analysis with ImageJ software.
Fig 4.
The alteration of ROS in normal liver cells and hepatoma cells.
(A,B,C) The level of ROS changes in QGY7701, HL7702 and SMMC7721 cells after treatment with DHM and NDP were detected using the DCFH assay. (B) The morphological changes in the mitochondria in QGY7701, HL7702 and SMMC7721 cells after treatment with the drug individually and in combination were detected using mito-tracker green. Each experiment was repeated at least three times.
Fig 5.
p53/Bcl-2 pathway was implicated in DHM and NDP induced dysfunction of mitochondria.
(A, B) p53-siRNA was used to knockdown of p53 and rescued the upregulation of p53 in hepatoma cells after DHM and NDP treatment. (C) Knockdown of p53 inhibited the downregulation of Bcl-2/Bax ratio caused by DHM and NDP treatment. (D) After si-RNA interference was performed, the morphological alteration of mitochondria in QGY7701, HL7702 and SMMC7721 cells after treatment with the drug individually and in combination were detected by using mito-tracker green.