Doxorubicin Attenuates Free Fatty Acid-Induced Lipid Accumulation via Stimulation of p53 in HepG2 Cells

Abstract

Non-alcoholic fatty liver disease (NAFLD) is characterized by excessive accumulation of fat in the liver, and there is a global increase in its incidence owing to changes in lifestyle and diet. Recent findings suggest that p53 is involved in the development of non-alcoholic fatty liver disease; however, the association between p53 expression and the disease remains unclear. Doxorubicin, an anticancer agent, increases the expression of p53. Therefore, this study aimed to investigate the role of doxorubicin-induced p53 upregulation in free fatty acid (FFA)-induced intracellular lipid accumulation. HepG2 cells were pretreated with 0.5 μg/mL of doxorubicin for 12 h, followed by treatment with FFA (0.5 mM) for 24 h to induce steatosis. Doxorubicin pretreatment upregulated p53 expression and downregulated the expression of endoplasmic reticulum stress- and lipid synthesis-associated genes in the FFA -treated HepG2 cells. Additionally, doxorubicin treatment upregulated the expression of AMP-activated protein kinase, a key modulator of lipid metabolism. Notably, siRNA-targeted p53 knockdown reversed the effects of doxorubicin in HepG2 cells. Moreover, doxorubicin treatment suppressed FFA -induced lipid accumulation in HepG2 spheroids. Conclusively, these results suggest that doxorubicin possesses potential application for the regulation of lipid metabolism by enhance the expression of p53 an in vitro NAFLD model.

Keywords: AMP-activated protein kinase; Doxorubicin; Fatty acid synthesis; Lipid metabolism; Non-alcoholic fatty liver disease; p53.

Fig. 1

Effect of doxorubicin on p53 expression and free fatty acid (FFA)-induced intracellular lipid accumulation in HepG2 cells. (A) Effect of doxorubicin on cell viability. For MTT assay, cells were treated with indicated concentration of doxorubicin for 48 h. (B) Doxorubicin caused a dose-dependent increase in p53 gene expression. (C) Doxorubicin enhanced the protein expression of p53. (D) Intracellular TG content. (E) Oil Red O staining was performed to assess lipid accumulation. TG analysis and Oil Red O intensity were quantified by total cell number and the data are presented from three independent experiments. (F) Intracellular localization of p53 and lipid accumulation in doxorubicin-treated cells. Nuclear staining was performed using DAPI (4’,6-diamidino-2-phenylindole). HepG2 cells were pretreated with 0.5 μg/mL of doxorubicin for 12 h, followed by incubation with 0.5 mM of FFA composed of BSA-conjugated oleic acid and palmitic acid (2:1) for 24 h. Data are presented as mean ± standard deviation (SD). ***p<0.001 (compared with CON, Student’s t-test). Different letters indicate significant differences (p<0.05) among the groups (one-way ANOVA followed by Newman-Keuls multiple comparison test). CON, control; Dox, doxorubicin.

Fig. 2

Doxorubicin reduces free fatty acid (FFA)-induced endoplasmic reticulum (ER) stress and reactive oxygen species (ROS) generation in HepG2 cells. (A) Gene expression of ER-stress associated factors were measured. (B) ROS signal was measured using DCFDA and quantified by total cell numbers. Cells were pretreated with doxorubicin for 12 h, followed by FFA treatment for 24 h. Data are presented mean ± standard deviation of values from three independent experiments. Different letters indicate significant differences (p<0.05) among the groups (one-way ANOVA followed by Newman-Keuls multiple comparison test). CON, control; Dox, doxorubicin.

Fig. 3

Doxorubicin regulates the expression of lipid synthesis- and export-associated genes in free fatty acid (FFA)-treated cells. Protein expression of (A) lipid synthesis and (B) lipid export associated factors after doxorubicin and/or FFA treatment. Cells were pretreated with doxorubicin for 12 h, followed by FFA treatment for 24 h. Data from three independent experiments are expressed as mean ± standard deviation (SD). Different letters indicate significant differences (p<0.05) among the groups (one-way ANOVA followed by Newman-Keuls multiple comparison test). CON, control; Dox, doxorubicin.

Fig. 4

p53 knockdown rescues the inhibitory effect of doxorubicin on free fatty acid (FFA)-induced lipid accumulation in HepG2 cells. (A) Protein expression of p53. (B) Intracellular triglyceride (TG) content and (C) Oil Red O staining. (D) Immunofluorescence staining for p53 and lipid content (PC6S). Nuclear staining was performed using DAPI. Cells were pretreated with doxorubicin for 12 h to induce p53, followed by transfection with p53 siRNA oligonucleotides for 12 h using Lipofectamine 3000. The medium was replaced with a medium containing 0.5 mM of FFA. Data from three independent experiments are presented mean ± standard deviation (SD). ***p<0.001 (Student’s t-test). Dox, doxorubicin.

Fig. 5

p53 knockdown reverses the effects of doxorubicin in free fatty acid (FFA)-treated HepG2 cells. (A) Gene expression of CHOP, SREBP-1, and PPARα. (B) Protein expression of endoplasmic reticulum (ER) stress (ATF6), lipid uptake (CD36), lipid synthesis (SREBP-1, FAS, SCD1), and AMPK-mediated lipid metabolism pathway (PPARα, AMPK, p-AMPK, ACC, p-ACC) in p53 knockdown cells after doxorubicin and FFA treatment. (C) Intracellular reactive oxygen species (ROS) level was assessed using DCFDA. ROS signal was quantified by total cell numbers. Cells were pretreated with doxorubicin for 12 h to induce p53, followed by transfection with p53 siRNA oligonucleotides for 12 h using Lipofectamine 3000. The medium was replaced with a medium containing 0.5 mM of FFA. Data from three independent experiments are expressed as mean ± standard deviation (SD). **^,***p<0.01 and 0.001 (Student’s t-test). Dox, doxorubicin.

Fig. 6

Doxorubicin downregulates free fatty acid (FFA)-mediated lipid accumulation in HepG2 3D spheroids. Lipid accumulation increased in a (A) FFA dose-dependent manner, (B) which was suppressed by doxorubicin treatment. HepG2 cells were cultured in PAMCELL^TM R100 3D 96-well plate until cell’s self-assembled spheroids formed. Cells were treated with doxorubicin for 12 h, followed by treatment with indicated concentrations of FFA. Lipid accumulation was assessed using PC6S. Relative intensity of each samples were measured using Image Studio software and each value represents the mean ± standard deviation (SD). ***p<0.001 (compared with 0 mM FFA, Student’s t-test). Different letters indicate significant differences (p<0.05) among the groups (one-way ANOVA followed by Newman-Keuls multiple comparison test). Dox, doxorubicin.