Emodin Induced SREBP1-Dependent and SREBP1-Independent Apoptosis in Hepatocellular Carcinoma Cells

Figures

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  Figure 1. Construction of knockout (KO) and overexpression (OE) cell lines. (A) The sequencing results of KO positive clone cells; (B) The messenger RNA (mRNA) level of SREBP1 in wild type (WT) and OE Bel-7402 cells. Data were expressed as mean ± standard error of the mean. ***P < 0.001 vs. WT group; (C and D) The protein level of SREBP1 in WT, OE, and KO Bel-7402 cells. Data were expressed as mean ± standard error of the mean. ***P < 0.001 vs. WT group.

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  Figure 2. Emodin inhibited cell proliferation and induced intrinsic apoptosis in Bel-7402 cells. (A) The chemical structure of emodin. (B) Bel-7402 cell viability after treatment with emodin determined by the MTT assay. (C) Apoptosis in emodin-treated Bel-7402 cells as assessed by flow cytometry. (D and E) The fluorescence images with JC-10 staining in Bel-7402 cells. Bel-7402 cells were exposed to emodin (100 μmol/L) for 24 h followed by 30-min incubation with JC-10. When the mitochondrial membrane potential (ΔΨm) was higher, the JC-10 accumulated in the mitochondria matrix to form the polymer producing red fluorescence; otherwise, the green fluorescence from the JC-10 monomers was used to represent the cells that lost ΔΨm. Quantitative data of the ratio of red and green fluorescence intensity were measured. (F and G) Expression level of intrinsic apoptosis-associated proteins was measured after emodin treatment by Western blotting analysis. Data were expressed as mean ± standard error of the mean. *P < 0.05, **P < 0.01, and ***P < 0.001 vs. ctrl group (0 μmol/L), respectively.

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  Figure 3. Emodin decreased lipid metabolism in Bel-7402 cells. (A) Total cellular TG content of Bel-7402 cells treated with emodin. (B) High-performance liquid chromatography-mass spectrometry (HPLC-MS) determination of the saturated fatty acids (SFAs) to monounsaturated fatty acids (MUFAs) ratios (palmitic acid to palmitoleic acid, 16:0 to 16:1) (stearic acid to oleic acid, 18:0 to 18:1) as well as the content of 16:0, 16:1, 18:0, and 18:1 in Bel-7402 cells treated with emodin. (C) The transcript level of lipid-associated genes in Bel-7402 cells when exposed to emodin. (D and E) The protein level of lipid-associated genes in Bel-7402 cells when exposed to emodin. Data were expressed as mean ± standard error of the mean. *P < 0.05, **P < 0.01, and ***P < 0.001 vs. ctrl group, respectively.

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  Figure 4. KO and OE of SREBP1 influenced lipid metabolism in Bel-7402 cells. (A) Total cellular TG content of SREBP1-KO, SREBP1-OE, and WT Bel-7402 cells. (B) HPLC-MS determination of the SFAs to MUFAs ratios (palmitic acid to palmitoleic acid, 16:0 to 16:1) (stearic acid to oleic acid, 18:0 to 18:1) as well as the content of 16:0, 16:1, 18:0, and 18:1 in SREBP1-KO, SREBP1-OE, and WT Bel-7402 cells. (C) The transcript level of lipid-associated genes in SREBP1-KO, SREBP1-OE, and WT Bel-7402 cells. (D and E) The protein level of lipid-associated genes in SREBP1-KO, SREBP1-OE, and WT Bel-7402 cells. Data were expressed as mean ± standard error of the mean. *P < 0.05, **P < 0.01, and ***P < 0.001 vs. WT group, respectively.

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  Figure 5. KO and OE of SREBP1 influenced intrinsic apoptosis in Bel-7402 cells. (A) Apoptotic cells were detected by the PE Annexin V-7AAD double staining assay in SREBP1-KO, SREBP1-OE, and WT Bel-7402 cells. (B and C) The ΔΨm was detected by JC-10 assay in SREBP1-KO, SREBP1-OE, and WT Bel-7402 cells. (D and E) The expression of intrinsic apoptosis associated-proteins in SREBP1-KO, SREBP1-OE, and WT Bel-7402 cells. Data were expressed as mean ± standard error of the mean. *P < 0.05, **P < 0.01, and ***P < 0.001 vs. WT group, respectively.

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  Figure 6. Emodin induced SREBP1-dependent and -independent apoptosis. (A) Apoptotic cells were detected using the PE Annexin V-7AAD double staining assay in SREBP1-KO and WT Bel-7402 cells after treatment with emodin. (B and C) The ΔΨm was detected using JC-10 assay in SREBP1-KO and WT Bel-7402 cells. Data were expressed as mean ± standard error of the mean. ***P < 0.001 vs. WT group, respectively. ### P < 0.001 vs. SREBP1-KO group, respectively.

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  Figure 7. Emodin activated apoptosis-associated proteins by blocking in a SREBP1-dependent and SREBP1-independent manner. (A and B) The expression of intrinsic apoptosis-associated proteins in SREBP1-KO and WT Bel-7402 cells. Data were expressed as mean ± standard error of the mean. *P < 0.05, **P < 0.01, and ***P < 0.001 vs. WT group, respectively. # P < 0.05, ## P < 0.01, and ### P < 0.001 vs. KO group, respectively.