Figure 4.

SCT‐induced excessive lipid biosynthesis is responsible for cell senescence and suppression in tumor cells. A) Increased gene expression levels of key enzymes in cholesterol synthesis (HMGCR, HMGCS1, SQLE, and IDI1), as well as fatty acid oxidation (CPT‐1) and synthesis (ACC1 and FASN) in tumor cells were induced with SCT treatment for 24 h. Total RNA was isolated from the treated tumor cells and gene expression was analyzed by real‐time qPCR. Expression levels of each gene were normalized to β‐actin expression level and adjusted to the levels in tumor cells treated with medium only (served as 1). Data shown are mean ± SD from three independent experiments with similar results. *p<0.05 and **p<0.01, compared with the medium‐only group. B) Accumulated LDs in tumor cells induced by SCT treatment. MCF7 and HCT116 cells were cultured in the presence of indicated concentrations of SCT for 24 or 48 h. The treated tumor cells were performed Oil Red O staining. The Oil Red O ⁺ tumor cells were identified with red granules as indicated by the arrows. Data shown in the right panels are means ± SD from three independent experiments. **p<0.01, compared with the medium‐only group. Scale bar: 30 µm. C) Schematic diagram of the lipid biosynthesis pathways. The key enzymes are shown in blue color and the specific pharmacological inhibitors used in this study are shown in red color. D) SCT treatment upregulated gene expression of key enzymes (ACAT1, ACAT2, and cPLA2α) involved in LD formation, but not hydrolase LIPA in tumor cells. MCF7 and HCT116 cells were treated with SCT (5 mm) for 24 h and mRNA expression levels of each gene were determined by the real‐time qPCR. The expression levels were normalized to β‐actin expression and adjusted to the levels in tumor cells with medium only. Data are mean ± SD from three independent experiments with similar results. *p<0.05 and **p<0.01, compared with the tumor cells in the respective medium only group. E) Blockage of the lipid synthesis reversed SCT‐induced LD accumulation in tumor cells. MCF7 and HCT116 cells were pretreated with the pharmaceutical inhibitors for lipid synthesis for 24 h, including C75 (5 µm), simvastatin (1 µm), 25‐HC (0.25 µg mL⁻¹), or avasimible (1 µm), respectively. Tumor cells were then cultured in the presence of SCT (5 mm) for an additional 24 h and stained for Oil Red O. Data shown in the right panels are mean ± SD from three independent experiments with similar results. *p<0.05 and **p<0.01, compared with the medium‐only group. ^## p<0.01, compared with the citrate treatment only group. Scale bar: 30 µm. F,G) Inhibition of the lipid synthesis blocked citrate‐induced suppression on tumor cell proliferation and growth. Cell treatment and procedure were identical to (E). Cell proliferation and growth were determined with the MTT assay (in F) and cell numbers counting (in G), respectively. Proliferation of tumor cells with medium only served as 100% (in F). Data shown are mean ± SD from three independent experiments. **p<0.01, compared with the medium‐only group. ^# p<0.05 and ^## p<0.01, compared with the citrate treatment only group. H) Blockage of the lipid synthesis prevented citrate‐induced tumor cell senescence. Cell treatment and procedure were identical to (E). Senescent cell populations were determined using the SA‐β‐Gal staining. Data shown in histograms are mean ± SD from three independent experiments. **p<0.01, compared with the medium‐only group. ^## p<0.01, compared with the citrate treatment only group. Scale bar: 30 µm. ANOVA was performed in (A–H).