Antiproliferative Effect of 7-Ketositosterol in Breast and Liver Cancer Cells: Possible Impact on Ceramide, Extracellular Signal-Regulated Kinases, and Nuclear Factor Kappa B Signaling Pathways

Abstract

Background: This study aimed to examine the effect of 7-Ketositosterol (7-KSS), on sphingomyelin/ceramide metabolites and apoptosis in human breast MCF-7 and human liver HepG2 cancer cells. Methods: Anti-proliferative effects of 7-KSS treatment were assessed at different concentrations and periods. Cell viability was assessed through MTT analysis, whereas the levels of sphingosine-1-phosphate (S1P), sphingomyelins (SMs), and ceramides (CERs) were measured using LC-MS/MS. Phosphorylated 44/42 ERK1/2 and NF-κB p65 (Ser536) protein levels were measured by Western blot analysis and immunofluorescence staining. Apoptosis was evaluated by TUNEL staining and flow cytometric assessment of annexin-V and propidium iodide (PI) labeling. Results: Treatment with 7-KSS significantly decreased cell survival and S1P, p-44/42 ERK1/2, and p-NF-κB p65 protein levels in cancer cells compared to controls. A substantial rise was detected in intracellular amounts of C16-C24 CERs and apoptosis in cancer cells incubated with 7-KSS. Conclusions: 7-KSS stimulated ceramide accumulation and apoptosis while decreasing cell proliferation via downregulating S1P, p-44/42 ERK1/2, and p-NF-κB p65 protein levels.

Keywords: 7-Ketositosterol; ERK; apoptosis; ceramide.

Figure 1

Analysis of 7-Ketositosterol toxicity in cancer cells and BJ fibroblasts. (A) 24 h viability analysis of 7-KSS administration in MCF-7 cells. Values mean ± SD (n = 8). One-way ANOVA or Kruskal–Wallis analysis was performed for statistical testing. The difference between the groups was analyzed by Tukey or Dunn’s test. *, p < 0.05 vs. control, DMSO, and 5 μM 7-KSS groups over the same time periods. #, p < 0.05 compared to 12 h and 18 h in the same dose interval. (B) 24 h viability analysis of 7-KSS administration in HepG2 cells. Values mean ± SD (n = 8). One-way ANOVA or Kruskal–Wallis analysis was performed for statistical testing. The difference between the groups was analyzed by Tukey or Dunn’s test. *, p < 0.05 vs. control, DMSO, 5 μM 7-KSS, and 10 μM 7-KSS over the same time periods. #, p < 0.05 compared to 12 and 18 h in the same dose range. (C) 24 h viability analysis of 7-KSS administration in BJ fibroblast cells. Values mean ± SD (n = 7–8). One-way ANOVA or Kruskal–Wallis analysis was performed for statistical testing. The difference among the groups was analyzed by Tukey or Dunn’s test. #, p < 0.05 vs. 18 h at the same dose.

Figure 2

(A) Representative Immunofluorescent staining of p-ERKs, p-NF-κB, and PCNA in MCF-7 cells incubated with 30 µM 7-Ketositosterol for 24 h. A 40× objective lens was used to obtain double-labeled images. (B) Quantitation of p-ERK fluorescence staining by ImageJ software. Values mean ± SD (n = 10). Kruskal–Wallis test and Dunn’s multiple comparison analysis were performed to test statistical significance. * p < 0.05 vs. control and DMSO groups. (C) Quantitation of p-NF-κB fluorescence staining. Values are given as mean ± SD (n = 10). One-way ANOVA test and Tukey’s multiple comparisons were used to determine statistical significance. * p < 0.05 compared to control and DMSO. (D) PCNA staining quantitation. Values are given as mean ± SD (n = 10). One-way ANOVA test and Tukey’s multiple comparisons were used to determine statistical significance. * p < 0.05 compared to control and DMSO. (E) PCNA levels in MCF-7 cells. Values mean ± SD (n = 5). *, p < 0.05 vs. control and DMSO groups.

Figure 3

(A) Representative immunofluorescent staining of p-ERKs, p-NF-κB, and PCNA in HepG2 cells incubated with 30 µM 7-Ketositosterol for 24 h. A 40× objective lens was used to obtain double-labeled images. (B) Quantitation of p-ERK fluorescence staining by ImageJ software. Values mean ± SD (n = 10). Kruskal–Wallis test and Dunn’s multiple comparison analysis were performed to test statistical significance. * p < 0.05 vs. control group. (C) Quantitation of p-NF-κB fluorescence staining. Values are given as mean ± SD (n = 10). One-way ANOVA test and Tukey’s multiple comparisons analysis were used for statistical analysis. * p < 0.05 compared to control and DMSO groups. (D) Quantitation of PCNA fluorescence staining by ImageJ software. Values mean ± SD (n = 10). Kruskal–Wallis test and Dunn’s multiple comparison analysis were performed to test statistical significance. * p < 0.05 vs. control and DMSO groups. (E) PCNA levels in HepG2 cell groups. Values mean ± SD (n = 5). One-way ANOVA and Tukey multiple comparison test were performed for statistical analysis. *, p < 0.05 vs. control and DMSO groups.

Figure 4

(A) Illustrative Western immunoblots for phospho-44/42 ERK1/2 (T202/Y204), total ERK 1/2, phospho-NF-κB p65 (S536), total NF-κB p65, and β-Actin in MCF-7 and HepG2 cells. C, control; D, cells treated with 1 μL/mL dimethyl sulfoxide; 7-KSS, cell treated with 30 μM 7-KSS for 24 h. (B) The band quantitation ratio of phospho-ERK/Total ERK in MCF-7 and (C) HepG2 cells. (D) The band quantitation ratio of phospho-NF-κB p65/Total NF-κB p65 in MCF-7 and (E) HepG2 cells. Blots were quantitated by ImageJ software. Data shown are representative of 3 separate experiments and values are given as mean ± SD. Statistical analysis was performed by one-way ANOVA and all pairwise multiple comparison procedures carried out by the Tukey test. *, p < 0.01 vs. control and DMSO groups. (F) Phospho-ERK levels in MCF-7 cells. Values mean ± SD (n = 5–8). One-way ANOVA and Tukey multiple comparisons were used to determine statistical significance. *, p < 0.05 vs. control and DMSO groups. (G) Phospho-ERK levels in HepG2 cells. Values mean ± SD (n = 8). One-way ANOVA and Tukey multiple comparisons were performed for statistical analysis. *, p < 0.05 when compared with the control and DMSO groups. (H) Phospho-NF-κB p65 levels in MCF-7 cells. Values mean ± SD (n = 8). One-way ANOVA and Tukey multiple comparisons were performed to test statistical significance. *, p < 0.05 when compared with the control and DMSO groups. (I) Phospho-NF-κB p65 levels in HepG2 cells. Values mean ± SD (n = 8). One-way ANOVA and Tukey multiple comparisons were used to determine statistical significance. *, p < 0.05 when compared with the control and DMSO groups.

Figure 5

TUNEL staining in cancer cells. (A) Dimethyl sulfoxide (DMSO, 1 μL/mL) and 7-KSS (30 μM) were applied for 24 h (200× magnification). (B) Quantitation of TUNEL staining in MCF-7 cells with the ImageJ program. Values mean ± SD (n = 7–8). Kruskal–Wallis test and Dunn’s analysis were performed to test statistical significance. *, p < 0.05 compared with the control and DMSO groups. (C) Quantitation of TUNEL staining in HepG2 cells with ImageJ program. Values are mean ± SD (n = 8). One-way ANOVA and Tukey multiple comparisons were used to determine statistical significance. *, p < 0.05 when compared with the control and DMSO groups. (D) Annexin V–FITC and PI-labeled HepG2 and MCF-7 cells. In total, 10,000 events were analyzed for each condition. (E) Quantitative analysis of annexin-V and PI labeling in MCF-7 cells by flow cytometry. Values mean ± SD (n = 3). One-way ANOVA analysis and Tukey test were used for statistical analysis. *, p < 0.05 compared to control and DMSO groups. (F) Quantitative analysis of annexin-V and PI labeling in HepG2 cells by flow cytometry. Values mean ± SD (n = 3). One-way ANOVA analysis and Tukey test were used for statistical analysis. *, p < 0.05 compared to control and DMSO groups.