Figure 1.
Effects of different concentrations of capsaicin on HepG2 cell viability.
HepG2 cells were treated with capsaicin from 0.05 to 2 mM for 24 and 48 h. Data considered as the control were defined as cells treated with a medium or vehicles (0.1% DMSO) without capsaicin. (A) Effect of capsaicin on the proliferation of HepG2 cells was expressed as percentage of cell viability compared with 100% of the control. Effects of 24 (B) and 48 (C) h exposure to varying concentrations of capsaicin on cell proliferation are expressed as concentrations of capsaicin at which the viability of cells can be reduced to 50% (IC50). Data from at least three independent experiments performed in at least triplicates are presented as means ± SD, n = 9, *p<0.05.
Figure 2.
The effect of capsaicin on the rate of apoptosis on HepG2 cells.
HepG2 cells were treated with 0.05, 0.2, and 0.5 mM of capsaicin for 24 h (A, B) and 48 h (C, D). The above panel shows a dual parametric dot plot (A, C) of cell population detection by flow cytometry depicting the distribution of viable, early, late apoptotic, and nec rotic cells evaluated by dual staining of PI fluorescence expressed on the y-axis versus Alex flour-Annexin V fluorescence expressed on the x-axis. The percentages (B, D) of cell populations relative to the whole cell populations (set as 100%) were expressed by bar charts showing the proportion of viable, early, and late apoptotic cells. The control was defined as cells treated with a medium or vehicles without capsaicin. Data from at least three independent experiments performed in at least triplicates are presented as means ± SD, n = 7.
Figure 3.
The effect of capsaicin on nuclear morphological alterations in HepG2 cells.
HepG2 cells were incubated with capsaicin at concentrations of 0.05 mM for 0, 6, 24 and 48 h. DAPI dye was used to indicate the apoptotic morphological features, such as chromatin condensation, nuclear fragmentation, and membrane blebbing, and then visualized by fluorescence microscopy at 40x magnifications. The control was defined as cells treated with a medium or vehicles without capsaicin (Scale bar: 25 µm).
Figure 4.
The effect of capsaicin on cell cycle distribution in HepG2 cells.
HepG2 cells were incubated with 0.05, 0.2, and 0.5 mM of capsaicin for 24 h (A, B) and 48 h (C, D). The above panel (A, C) shows the histogram profile of cell cycle distribution in G0/G1, S, and G2/M phases detected by flow cytometry using PI staining. The bar chart (B, D) shows the percentage of cell populations in each phase of the cell cycle relative to the whole cell populations (set as 100%). The control was defined as cells treated with a medium or vehicles without capsaicin. Data are expressed as means ± SD from at least three separate experiments performed in triplicate, n = 3.
Figure 5.
Effect of capsaicin on the expression of FASN, ACC, and ACLY protein levels in HepG2 cells.
HepG2 cells were incubated with 0.05, 0.2, and 0.5 mM of capsaicin for 24 h (A, B) and 48 h (C, D), and then the cell lysates were subjected to the SDS-PAGE system. Protein expressions were detected by specific antibodies and. β-actin was used as an internal standard to confirm the integrity and equal protein loading. The bar graph (A, C) shows the quantification of band intensity as a ratio of the individual band intensity relative to the β-actin band intensity in the same blot (e.g. FASN/β-actin). The band (B, D) results shown are representative of those obtained from at least three independent experiments. Data are expressed as means ± SD from at least three separate experiments performed in triplicate, n = 5, *p<0.05.
Figure 6.
Effects of capsaicin on fatty acid synthesis in HepG2 cells.
Intracellular long chain fatty acid (A) and intracellular triglyceride (B) synthesis were determined by using the free fatty acid and triglyceride quantification kits. HepG2 cells were incubated with 0.5 and 1.0 mM of capsaicin for 24 h. The control was defined as cells treated with a medium or vehicles without capsaicin. Data from at least three independent experiments performed in triplicate are represented as percentage of individual value in nmol/L compared to 100% of control values and are shown as means ± SD, n = 3, *p<0.05.
Figure 7.
Effect of capsaicin on induction of mitochondrial dependent apoptosis in HepG2 cells.
HepG2 cells were incubated with 0.5 mM capsaicin for 3 to 24 h. (A) The ΔΨm was examined by 2 µM JC-1 dye and detected by flow cytometry. The control was defined as cells treated with a medium or 0.1% DMSO vehicle without capsaicin. Histograms show the percentage of an energized and high polarization mitochondrial state as calculated by a ratio of red and green fluorescence intensity compared to the control. The decrease in percentage of ΔΨm represented a decrease in the red to green fluorescence intensity ratio. CCCP was used as a positive control to induce disruption of ΔΨm. (B) The effect of capsaicin on the rate of apoptosis on HepG2 cells following 0.5 mM capsaicin treatment for 3 to 24 h. The percentages of cell populations were expressed as bar charts showing the proportion of viable and apoptotic cells detected by flow cytometry using dual staining of PI and Alex flour-Annexin V fluorescence. Data from at least three independent experiments performed in at least triplicates are presented as means ± SD, n = 3.
Figure 8.
Effects of C75 on FASN protein expression, free fatty acid levels, triglyceride levels, and disruption of ΔΨm in HepG2 cells.
HepG2 cells were treated with C75 at 0.1 mM concentration for 6 h and 24 h. The control was defined as cells treated with a medium or vehicles without C75. (A) For the effect of capsaicin on the expression of FASN protein level in HepG2 cells, β-actin was used as the internal standard. The bar graph shows the quantification of band intensity as a ratio of FASN protein band intensity relative to the β-actin band intensity in the same blot (e.g. FASN/β-actin). For the effect of capsaicin on intracellular long chain fatty acid (B) and triglyceride synthesis (C) were determined by using the free fatty acid and triglyceride quantification kits, data are represented as percentage of the individual value in nmol/L compared to 100% of control values. (D) The bar chart shows the percentage of disruption of ΔΨm compared to the control (set as 100%). The change in ΔΨm was determined by staining cells with JC-1 fluorescence dye and detected by flow cytometry. Data from at least three independent experiments performed in triplicate are shown as means ± SD, n = 3, *p<0.05.
Figure 9.
Effect of capsaicin on the expression of FASN, ACC, and ACLY protein levels in HepG2 cells.
HepG2 cells were incubated with 0.05, 0.2, and 0.5 mM of capsaicin for 3 h (A, B), 6 h (C, D), and 12 h (E, F). (G) Histograms show the time-course effect of 0.5 mM capsaicin on protein expression in HepG2 cells for 3 to 48 h. Equal amounts of total protein were subjected to immunoblotting analysis with specific antibodies. β-actin was used as the internal standard to confirm the integrity and equal protein loading. Immunoreactive bands shown are representative of those obtained from at least three independent experiments. The bar graph shows the quantification of band intensity as a ratio of the individual band intensity relative to the β-actin band intensity in the same blot (e.g. FASN/β-actin). (H) Intracellular long chain fatty acid and triglyceride synthesis were determined by using the free fatty acid and triglyceride quantification kits following 0.5 mM capsaicin treatment for 3 h. The control was defined as cells treated with a medium or vehicles without capsaicin. Data are expressed as means ± SD from at least three separate experiments performed in triplicate, n = 3, *p<0.05.
Figure 10.
Effect of capsaicin on ROS generation in HepG2 cells.
HepG2 cells were treated with 0.5 mM of capsaicin for 2, 3, and 12 h. The control was defined as cells treated with a medium or 0.1% DMSO vehicle without capsaicin. ROS production was measured by CM-H2DCFDA fluorescent dye and detected by flow cytometry. Representative histogram plots using flow cytometric analysis indicate percentage of ROS generation compared to the control group (100%). ROS production following 0.1 mM C75 treatment for 12 h was measured. Data are expressed as means ± SD from at least three separate experiments performed in triplicate, n = 3, *p<0.05.
Figure 11.
An accumulation of Malonyl-CoA plays a vital role in capsaicin-induced apoptosis in HepG2 cells.
HepG2 cells were treated with 0.5 mM capsaicin or 0.1 mM C75 or 10 µg/ml TOFA for 12 h. Co-incubation of capsaicin or C75 with TOFA was performed by pre-incubation TOFA 1 h before treatment cells with capsaicin or C75. The control was defined as cells treated with a medium or 0.1%DMSO vehicle alone. (A) The ΔΨm was examined by 2 µM JC-1 dye and detected by flow cytometry. Histograms show the percentage of an energized and high polarization mitochondrial state as calculated by a ratio of red and green fluorescence intensity compared to the control. The decrease in percentage of ΔΨm represented a decrease in the red to green fluorescence intensity ratio. CCCP at 0.05 mM was used as a positive control to induce disruption of ΔΨm. (B) ROS production was measured by CM-H2DCFDA fluorescent dye and detected by flow cytometry. Histogram plots using flow cytometric analysis indicates percentage of ROS generation compared to the control group (100%). Data are expressed as means ± SD from at least three separate experiments performed in triplicate, n = 3, *p<0.05.
Figure 12.
Effects of capsaicin on normal human hepatocytes compared with HepG2 cells.
Cells were treated with 0.5 mM concentration of capsaicin for 3 h or 6 has indicated. The control was defined as cells treated with a medium or vehicles without capsaicin. (A) The bar graph shows the quantification of band intensity as a ratio of FASN protein band intensity relative to the β-actin band intensity in the same blot (e.g. FASN/β-actin). (B) For the effect of capsaicin on intracellular long chain fatty acid and (C) triglyceride synthesis determined by using the free fatty acid and triglyceride quantification kit, data are represented as percentage of the individual value in nmol/µL compared to 100% of control values. (D) The bar chart shows the percentage of disruption of mitochondrial membrane potential relative to the control (set as 100%) after 6 h of capsaicin treatment by JC-1 fluorescence dye staining and detected by flow cytometry. Data from at least three independent experiments performed in triplicate are shown as means ± SD, n = 3, *p<0.05.