Fig 1.
Gyp potentiates 5-Fu-induced cell proliferation inhibition.
(A) Chemical dammarane skeleton structure of Gypenosides. (B) Cell viability was measured by MTT assay at 48 h after 5-Fu treatment in SW-480,SW-620 and Caco2 cells. (C-F) Cell viability was measured by MTT assay at 24 and 48 h after 5-Fu and / or Gyp treatment in SW-480,SW-620 and Caco2 cells. Combination index (CI) value was analyzed using CalcuSyn software. CI < 1 indicates a synergistic effect. (G) Colony formation of SW-480 and Caco2 cells after 5-Fu and / or Gyp treatment. All data are expressed as means ± SD of triplicates and p* < 0.05, p** < 0.01 versus control; p## < 0.01 versus 5-Fu or Gyp alone group.
Fig 2.
Apoptotic response induced by 5-Fu and Gyp.
(A) Cell apoptosis was detected by Annexin V-PE/7-AAD assay after 5-Fu and / or Gyp treatment for 24 h in SW-480 cells. (B) The expression level of caspase 3, caspase 9, Bcl-2 and cleaved-PARP in SW-480 cells was detected by western blotting. (C) Nuclear condensation and cell morphology change in SW-480 cells after 5-Fu co-treated with Gyp was observed using Hoechst 33342 staining. Experiments were done independently in triplicate per experimental point, and representative results were shown. All data are expressed as means ± SD of triplicates and p* < 0.05, p** < 0.01 versus control.
Fig 3.
Effects of 5-Fu and / or Gyp on cell cycle distribution.
(A) Cell cycle distribution after 5-Fu and Gyp treatment. (B) The expression of phospho-p53, phospho-cdk2 and cyclin E after 5-Fu and / or Gyp treatment in SW-480 cells were detected by western blotting. Experiments were done independently in triplicate per experimental point, and representative results were shown. All data are expressed as means ± SD of triplicates and p* < 0.05, p** < 0.01 versus control.
Fig 4.
DNA response induced by 5-Fu, Gyp and 5-Fu + Gyp.
(A) DNA fragmentation was detected using flow cytometry after 5-Fu, Gyp and 5-Fu + Gyp treatment in SW-480 cells after 24 and 48 h. Histograms showed number of cell channels (vertical axis) vs. PI fluorescence (horizontal axis). (B) Comet assay was performed for detection of DNA damage after 5-Fu co-treated with Gyp. (C) The expression level of Ser139-Histone H2A.X was determined using western blotting. Experiments were done independently in triplicate per experimental point, and representative results were shown. All data are expressed as means ± SD of triplicates and p* < 0.05, p** < 0.01 versus control; p## < 0.01 versus 5-Fu or Gyp alone group.
Fig 5.
ROS accumulation after 5-Fu + Gyp treatment.
(A) Intracellular ROS production after 5-Fu and / or Gyp treatment was detected using DCFH-DA staining. (B-D) Intracellular ROS production, DNA fragmentation and cell apoptosis in SW-480 cells were measured after added NAC to culture medium simultaneously with 5-Fu and Gyp. Experiments were done independently in triplicate per experimental point, and representative results were shown. All data are expressed as means ± SD of triplicates and p** < 0.01 versus control; p## < 0.01 versus 5-Fu + Gyp group.
Fig 6.
Role of p53 in 5-Fu + Gyp treatment.
(A) Cell viability, (B) Intracellular ROS production and (C) DNA damage were detected after added pifithrin-α to culture medium simultaneously with 5-Fu plus Gyp. Experiments were done independently in triplicate per experimental point, and representative results were shown. All data are expressed as means ± SD of triplicates and p** < 0.01 versus control; p## < 0.01 versus 5-Fu + Gyp group.
Fig 7.
Anti-tumor effect of 5-Fu and / or Gyp on CT-26-colon-carcinoma-bearing mice.
(A) The growth of tumor volume in different groups were monitored every day after inoculated with CT-26 cells for seven days. (B) The final tumor weight in each group after treatment. (C) The microstructure of tumor tissue in different groups was observed by Hematoxylin-eosin staining. (D) The expression level of PCNA after different treatment was performed using immunohistochemistry detection and quantified values shown are the average percentage of positive nuclei. All data are expressed as means ± SD of triplicates and p* < 0.05, p** < 0.01 versus control; p## < 0.01 versus 5-Fu or Gyp alone group.
Fig 8.
Toxicity evaluation of combination treatment.
(A) Body weights were recorded every day in different groups every day after inoculated with CT-26 cells for seven days. (B) Spleen indexes were calculated after various treatment. (C-F) The level of AST, ALT, BUN and Cr in the serum were detected. All data are expressed as means ± SD of triplicates.
Fig 9.
Proposed pathways of Gyp potentiates 5-Fu induced cell apoptosis and cell cycle arrest.