Figure 1.
Antiproliferative effect of fatty acid esters of phloridzin on HepG2 and normal cells.
Hepatic carcinoma (HepG2) cells and normal human hepatocytes (HP-F) and rat hepatocytes (RTCP10) cells were exposed to test compounds at 1, 10, 50, 100 µM for 24 h. The cell viability was determined using MTS assay. The data presented as the percentage viability relative to vehicle only treated control group. Data are presented as the mean ± SD (n = 3) are representative of at least three separate independent experiments. *P<0.05 significantly different from the vehicle only control group (Tukey HSD, P<0.01).
Table 1.
EC50± SD values of tested compounds in three human cancer cell lines and a normal cells (HP-F).
Figure 2.
Percentage LDH release relative to control in HepG2 cells.
The cells were incubated with 100 µM of fatty acid esters of phloridzin (Pz) in comparison with parent compounds phloridzin, phloretin (aglycone), or liver cancer drug sorafenib for 24 h. Data are presented as the mean ± SD (n = 3) are representative of at least three separate independent experiments. Different letters represent significantly different mean values from other treatments (Tukey HSD, P<0.01).
Figure 3.
Inverted phase contrast microscopy images of HepG2 cells.
The cells were cultured (2×104 cells/100 µL) in 96 well plate after incubation with 100 µM of fatty acid esters of phloridzin (Pz) in comparison with parent compounds phloridzin, phloretin (aglycone) or liver cancer drug sorafenib for 24 h. Shown are representative images of three independent experiments.
Figure 4.
Fluorescent microscopic analysis of apoptotic HepG-2 cells stained with Annexin V-FITC and 7-AAD.
The cells were cultures (1×105 cells) in chambered slides and incubated with 100 µM of fatty acid esters of phloridzin (Pz) in comparison with parent compounds phloridzin or phloretin (aglycone) or liver cancer drug sorafenib. Green and red color indicates early and late apoptotic cells, respectively. Shown are representative images of three independent experiments.
Figure 5.
DNA fragmentation of HepG2 cells.
DNA were isolated after incubation of HepG2 cells with 100 µM of fatty acid esters of phloridzin (Pz) in comparison with parent compounds phloridzin, free fatty acids, phloretin (aglycone) or liver cancer drug sorafenib for 24 h. Lane M: molecular-weight marker, lane C: control, lane 1–6: stearic acid ester of Pz, oleic acid ester of Pz, linoleic acid ester of Pz, α-linolenic acid ester of Pz, DHA ester of Pz and EPA ester of Pz, lane 7: sorafenib, lane 8–13: stearic acid, oleic acid, linoleic acid, α-linolenic acid, DHA and EPA, lane 14: phloridzin, and lane 15: phloretin. Shown are representative gel images of three independent experiments.
Figure 6.
Caspase 3 activity of HepG2 cells.
The cells were incubated with 100 µM of fatty acid esters of phloridzin (Pz) in comparison with parent compounds phloridzin, phloretin (aglycone) or standard drug sorafenib for 24 h. Data are presented as the mean ± SD (n = 3) are representative of at least three separate independent experiments. Different letters represent significantly different mean values from other treatments (Tukey HSD, P<0.01).
Figure 7.
Mitochondrial membrane potential (Δψm) of HepG2 cells measured by JC-1 fluorescence.
HepG2 cells were treated with 100 µM of fatty acid esters of phloridzin (Pz) in comparison with parent compounds phloridzin, individual fatty acids, phloretin (aglycone) or liver cancer drug sorafenib for 24 h. The fluorescence of JC-1 monomers was measured at em 535 nm and aggregates at em 590 nm. Data are presented as the mean ± SD (n = 3) are representative of at least three separate independent experiments. Different letters represent significantly different mean values from other treatments (Tukey HSD, P<0.01).
Figure 8.
Cellular ATP level in HepG2 cells.
The cells were treated with 100 µM of fatty acid esters of phloridzin (Pz) in comparison with parent compounds phloridzin, free fatty acids, phloretin (aglycone) or liver cancer drug sorafenib for 24 h. Cellular ATP content in treated cells is expressed as percentage compared to ATP levels in untreated controls. Data are presented as the mean ± SD (n = 3) are representative of at least three separate independent experiments. Different letters represent significantly different mean values from other treatments (Tukey HSD, P<0.01).
Figure 9.
Evaluation of cell cycle in HepG2 cells using the propidium iodide assay.
HepG2 cells were treated with 100 µM of fatty acid esters of phloridzin (Pz), pz or phloretin or sorafneib for 24 h, and then cell cycle profile was determined using flow cytometry after staining with PI/RNase. Representative FACS histograms of nuclear DNA content are shown (A). The DNA content peaks indicating cells in the G0G1, S, or G2/M phase of the cell cycle are marked. The bar chart show the changes in percentages (mean ± SD) of cells in G0/G1, S-phase, and G2/M after treatment (B). Data are presented as the mean ± SD (n = 3) are representative of three separate independent experiments. Different letters represent significantly different mean values from other treatments (Tukey HSD, P<0.01).
Figure 10.
Activity of human topoisomerase II of HepG2 cells.
The cells were incubated with 100 µM of fatty acid esters of phloridzin (Pz) in comparison with parent compounds phloridzin, phloretin (aglycone) or sorafenib for 24 h. A. Lanes 1 Pz-oleic acid, lane 2: Pz-stearic acid, lane 3: Pz-linoleic acid, lane 4: Pz-α-linolenic acid, lane 5: Pz-DHA, lane 6: Pz-EPA, lane 7: phloridzin, lane 8: sorafenib, and lane 9: phloretin. The data are representative of three separate, independent experiments. B. Percentage inhibition.
Table 2.
Genes with altered expressions in HepG2 cells treated with DHA ester of phloridzin or sorafenib.