| To study the effect of emodin on improving chemotherapeutic drug (5-fluorouracil, 5-Fu) induced apoptosis in human hepatoma cell line HepG2, and the potential mechanisms were discussed.We treated HepG2 cells with various concentrations of emodin for 2h, and also treated them with 4μg/ml emodin for different time. The dynamic changes of the intracellular ROS levels of HepG2 cells were detected by flow cytometry (FCM).MTT assay and FCM were used to assess cell viability, apoptosis and cell cycle distribution after treatment with various concentrations of emodin alone or combined with 5-Fu for different time. Transmission electron microscope was applied to observe the ultrastructural change of cells.Immunohistochemical staining and image analysis technique were used to analyze the changes of apoptosis-related proteins, including Bax, Bcl-2 and apoptosis-inducing factor (AIF) when HepG2 cells were treated with various concentrations of emodin alone or combined with 5-Fu for 24h. Terminal deoxynucleotidy1 transferase-mediated dUTP nick end labeling (TUNEL) was used to detect apoptosis of cells. Finally, the relationship between apoptosis and the expression of Bax, Bcl-2 and AIF proteins was analyzed.FCM result showed that emodin elevated intracellular ROS level in a time- and dose-dependent manner. Measurements using DCFH-DA demonstrated that incubation of HepG2 cells with 4μg/ml emodin for 0min, 15min, 30min, 1h, 2h, 4h, 8h, 16h or 24h led to a significant increase in the generation of ROS. The relative intracellular ROS levels were 42.67±0.80 (inherent cellular ROS level), 61.71±1.35, 66.64±1.44, 73.74±1.67, 88.87±1.81, 84.58±1.37, 63.34±1.84, 53.91±1.50 and 44.86±1.84, respectively. The level of ROS at 2h was about two-fold as the inherent level, and stayed at peak for 2h. Then, it began to decrease, the level of ROS at 24h was approximately as low as that of the inherent. The relative intracellular ROS levels of HepG2 cells that incubated in various concentrations of emodin (0μg/ml, 2μg/ml, 4μg/ml and 8μg/ml) for 2h were 46.15±1.46, 63.97±1.34, 73.90±1.69, 86.85±1.71, respectively.MTT assay demonstrated that emodin exhibited a statistically significant time- and dose-dependent growth-inhibitory effect on HepG2 cells which incubated in various groups: A group (the control group, medium alone), B group (10mg/l 5-Fu), C group (2μg/ml emodin), D group (4μg/ml emodin), E group (2μg/ml emodin + 10mg/l 5-Fu), F group (4μg/ml emodin + 10mg/l 5-Fu) for 4h, 8h, 16h or 24h. 5-Fu or emodin alone could inhibite the growth of cell, but both combined showed more obvious growth-inhibitory effect. Cell viability in any of the experiment groups was significantly decreased compared to that in the control group (P<0.05). Cell viability of E and F group was significant lower than that of B group (P<0.01), especially at 24h.FCM demonstrated that apoptotic cells (sub-G1 peak) could be observed obviously after treatment for 16h. The ratio of apoptotic cells after treated for 24h in B, C, D, E and F groups were (7.25±1.10) %, (11.71±1.36 )%, (12.98±1.67)%, (18.66±2.45)%, (27.27±2.63)%, respectively, it showed that the number of apoptotic cell elevated gradually with increase of concentrations of emodin. Cell fragments and necrosis increased significantly after incubated for 48h in F group, while the ratio of apoptotic cells decreased to (24.56±2.23)%. The proportions of cells in G1, S, and G2 were determined by FCM analysis, and cell cycle distribution in HepG2 cells was measured after treatment with various concentrations of emodin combined with 5-Fu for 24h (E group, F group). The DNA content analysis exhibited an increase of cell population in G1 phase, accompanied with decrease in S phase and G2 phase. This indicated a cell cycle was arrested at the G1 phase, and the effect was dose dependent (P<0.01).Electron microscopic was applied to observe the ultrastructural changes of HepG2 cells incubated for 24h in A～F groups. The result showed the most cells in control group were preserved in well ultrastructure, there were abundant organelles in cytoplasm and much microvillus on cell membrane, nucleus was round or irregular, nucleolus was obvious and euchromatin was predominant. Except some cells with well ultrastructure, some apoptotic cells were found in B, C and D groups. More apoptotic cells were found in E and F groups, especially in F group. The most cells in F group were at various stages of apoptosis. Apoptotic cell in early-stage showed a little cell shrinkage, microvillus decrease, chromatin margination, cytoplasm became denser. Apoptotic cell in mid-stage showed the volume of cell was more diminution, microvillus disappearance, chromatin was more margination and condensation. Apoptotic cell in late-stage showed nucleus pyknosis or breaking into discrete fragments, apoptotic bodies formed. There were some necrotic cells in F group: cell swollen, chromatinolysis, cell membrane destroyed and many denatured vacuoles in cytoplasm. There were some atypical apoptotic cells in E and F group with the change of nucleus similar as apoptosis, but cytoplasm swollen.Immunohistochemical staining were used to analyze the expression of apoptosis-related proteins including Bax, Bcl-2 and AIF when HepG2 cells were treated with various concentrations of emodin (2μg/ml, 4μg/ml) alone or combined with 5-Fu for 24h. It showed that the expression level of Bax protein in each experimental groups was significantly higher than that in control group; the expression levels of Bax protein in E and F groups were significantly higher than that in B group. The expression level of Bax protein in D group was significantly higher than that in C group (P<0.01), and F group higher than E group (P<0.01). The effect was dose dependent.The result of Immunohistochemical staining for Bcl-2 protein showed that the expression level of Bcl-2 protein in each experimental groups was significantly lower than that in control group; The expression level of Bcl-2 protein in D group was significantly lower than that in C group (P<0.01), and F group lower than E group (P<0.01). Compared with B group, the expression of Bcl-2 protein in E and F groups reduced significantly. The effect was dose dependent.Immunohistochemical staining result for AIF protein showed that the expression level of AIF protein in each experimental groups was significantly higher than that in control group; The expression level of AIF protein in D group was significantly higher than that in C group (P<0.01), and F group higher than E group (P<0.01). Compared with B group, the expression of AIF protein in E and F groups increased significantly. The effect was also dose dependent.TUNEL was used to detect apoptosis of HepG2 cells in A～F groups. It showed that the ratio of apoptotic cells of A～F groups is 3%, 10%, 14%, 18%, 21% and 31%, respectively. The ratio of apoptotic cells of each experimental groups was significantly higher than that of the control group (P<0.05). In F group, the ratio of apoptotic cells reached the highest level and there were some necrotic cells at the same time. The apoptotic response induced by emodin combined with 5-Fu in HepG2 cells was a dose-dependent manner, this was the similar as the result of FCM.We investigated the relationship between apoptosis and the expression of Bax , Bcl-2 and AIF proteins of HepG2 cells in A～F groups. It showed that the ratio of apoptotic cells was positively correlated with the expression of Bax protein (r=0.881, P<0.05), and so was the expression of AIF protein (r=0.900, P<0.05), while negatively correlated with the expression of Bcl-2 protein (r=-0.942, P<0.01).In summary, it would be concluded that emodin could elevate intracellular ROS levels of human hepatoma HepG2 cells in a dose- and time-dependent manner, and intracellular ROS levels of HepG2 cells incubated in 4μg/ml emodin for 2h reached the peak. Various concentrations of emodin combined with 5-Fu sensitized HepG2 cells to 5-Fu induced apoptosis via generation of ROS in a time- and dose-dependent manner. The ratio of apoptotic cells and expression levels of Bax and AIF proteins in the combination groups of emodin and 5-Fu were significantly higher than those of 5-Fu alone group, respectively, while the expression level of Bcl-2 protein was significantly reduced. Emodin could sensitize HepG2 cells to apoptosis induced by 5-Fu in a time- and dose-dependent manner. The synergic effect may be associated with the regulation of expression levels of Bax , Bcl-2 and AIF proteins. |
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