Atorvastatin Attenuates Programmed Death Ligand-1 (PD-L1) Induction in Human Hepatocellular Carcinoma Cells

Abstract

Liver cancer is the sixth most common cancer worldwide with high morbidity and mortality. Programmed death ligand 1 (PD-L1) is a major ligand of programmed death 1 receptor (PD1), and PD1/PD-L1 checkpoint acts as a negative regulator of the immune system. Cancers evade the host's immune defense via PD-L1 expression. This study aimed to investigate the effects of tumor-related cytokines, interferon gamma (IFNγ), and tumor necrosis factor alpha (TNFα) on PD-L1 expression in human hepatocellular carcinoma cells, HepG2. Furthermore, as atorvastatin, a cholesterol-lowering agent, is documented for its immunomodulatory properties, its effect on PD-L1 expression was investigated. In this study, through real-time RT-PCR, Western blot, and immunocytochemistry methods, PD-L1 expression in both mRNA and protein levels was found to be synergistically upregulated in HepG2 by a combination of IFNγ and TNFα, and STAT1 activation was mainly responsible for that synergistic effect. Next, atorvastatin can inhibit the induction of PD-L1 by either IFNγ alone or IFNγ/TNFα combination treatment in HepG2 cells. In conclusion, in HepG2 cells, expression of PD-L1 was augmented by cytokines in the tumor microenvironment, and the effect of atorvastatin on tumor immune response through inhibition of PD-L1 induction should be taken into consideration in cancer patients who have been prescribed atorvastatin.

Keywords: HepG2; IFNγ; PD-L1; TNFα; atorvastatin; liver cancer.

Figure 1

Individual or combination treatments of cytokines: IFNγ and/or TNFα are not toxic to HepG2 cells. HepG2 cells were treated with either IFNγ (A) or TNFα (B), or a combination of IFNγ and TNFα (C) at various concentrations (ng/mL) for 24 and 48 h and percentage of cell viability was assessed by MTT assays. Data are presented as mean ± SD, n = 3.

Figure 2

Combination treatment of IFNγ and TNFα synergistically upregulates PD-L1 expression both in mRNA and protein level in HepG2 cells. (A–D) Analysis of PD-L1 expression of HepG2 cells treated with either individual or combination of IFNγ and TNFα at various concentrations (ng/mL) for 6 h in RT-PCR analysis and for 24 h in Western blot or immunocytochemistry analysis. (A) Western blot analysis and quantification. β-actin was used as a loading control. (B) RT-PCR analysis. Relative PD-L1 mRNA level was normalized to the expression of the GAPDH gene. (C,D) Immunocytochemistry of PD-L1 membrane expression and its quantification. Scale bar = 50 μm. Data are presented as mean ±SD for WB and RT-PCR; mean ±SEM for Immunocytochemistry. n = 3. * p < 0.05, ** p < 0.01 compared to control. # p < 0.05, ## p < 0.01 compared to IFNγ single treatment.

Figure 3

Activation of signaling pathways by combination treatment of IFNγ and TNFα. (A–D) Western blot analysis and quantification of the phosphorylation status of signaling proteins in HepG2 cells induced by combination treatment of IFNγ 1 ng/mL and TNFα 10 ng/mL for 5, 15,30 and 60 min. (A) STAT1 and STAT3 (B) MAPK pathway: JNK, p38, and ERK (C) NFκB, and (D) Akt activation. The density ratio of proteins was shown as relative expression of phosphorylated form to respective total form. β-actin was used as a loading control. Data are presented as mean ± SD. n = 3. * p < 0.05 compared to control. # p < 0.05 compared to TNFα treatment of respective time points. $ p < 0.05 compared to IFNγ treatment of respective time point.

Figure 4

Enhanced upregulation of PD-L1 expression by combination treatment of IFNγ and TNFα compared to individual cytokine treatment is mainly dependent on STAT-1 signaling pathway. (A–C) Analysis of PD-L1 expression of HepG2 cells pre-treated for 2 h with individual or combination of inhibitors: JAK inhibitor (ruxolitinib 1 μM), JNK inhibitor (SP 600125 10 μM), NF-κB inhibitor (BAY 11-7082), which was followed by treatment with a combination of IFNγ 1 ng/mL and TNFα 10 ng/mL for 6 h in RT-PCR analysis, and for 24 h in Western blot analysis. (A,B) Western blot analysis and quantification. β-actin was used as a loading control. (C) RT-PCR analysis. Relative PD-L1 mRNA level was normalized to the expression of the GAPDH gene. Data are presented as mean ± SD. n = 3. * p < 0.05, ** p < 0.01 compared to control. # p < 0.05, ## p < 0.01 compared to IFNγ and TNFα combined treatment.

Figure 5

STAT-1 signaling is the major pathway for the upregulation of PD-L1 expression by combination treatment of IFNγ and TNFα. (A) Representative figure and (B) quantification of immunocytochemistry analysis of PD-L1 membrane expression in HepG2 cells pre-treated for 2 h with individual or combination of inhibitors: JAK inhibitor (ruxolitinib 1 μM), JNK inhibitor (SP 600125 10 μM), NF-κB inhibitor (BAY 11-7082), which was followed by treatment with a combination of IFNγ 1 ng/mL and TNFα 10 ng/mL for 24 h. Scale bar = 50 μm. Data are presented as mean ± SEM. n = 3. * p < 0.05, ** p < 0.01 compared to control. # p < 0.05, ## p < 0.01 compared to IFNγ and TNFα combined treatment.

Figure 6

Induction of PDL1 expression by IFNγ is diminished by Atorvastatin, STAT-1 inhibitor, in HepG2 cells. (A) MTT assay for the viability of HepG2 cells treated with various concentrations of atorvastatin (μM) for 24 or 48 h. (B,C) Analysis of the effect of atorvastatin on phosphorylation of STAT-1 protein in HepG2 cells pre-treated with atorvastain 10 μM for 4 h, followed by induction with IFNγ 20 ng/mL for 15, 30, 60 min by western blot. The density ratio of protein was shown as relative expression of phosphorylated form to the total form of STAT1. β-actin was used as a loading control. (D–F) Analysis of PD-L1 expression of HepG2 cells co-treated with IFNγ 20 ng/mL and atorvastatin at various concentrations (μM) for 6 h in RT-PCR analysis, and for 24 h in western blot analysis. (D,E) Western blot analysis and quantification. β-actin was used as a loading control. (F) RT-PCR analysis. Relative PD-L1 mRNA level was normalized to the expression of the GAPDH gene. Data are presented as mean ± SD. n = 3. * p < 0.05, ** p < 0.01 compared to control. # p < 0.05, ## p < 0.01 compared to IFNγ treatment.

Figure 7

Atorvastatin mitigate the effect of IFNγ on upregulation of PD-L1 expression in HepG2 cells. (A) Representative figure and (B) quantification of immunocytochemistry analysis of PD-L1 membrane expression in HepG2 cells co-treated with IFNγ 20 ng/mL and atorvastatin at various concentrations (μM) for 24 h. Scale bar = 50 μm. Data are presented as mean ± SEM. n = 3. * p < 0.05, ** p < 0.01 compared to control. # p < 0.05, ## p < 0.01 compared to IFNγ treatment.

Figure 8

Atorvastatin reduces the induction of PD-L1 expression by IFNγ and TNFα combined treatment in HepG2 cells. (A–D) Analysis of PD-L1 expression of HepG2 cells co-treated with a combination of IFNγ 1 ng/mL+TNFα 10 ng/mL and atorvastatin at various concentrations (μM) for 6 h in RT-PCR analysis, and for 24 h in western blot and immunocytochemistry analysis. (A) Western blot analysis and quantification. β-actin was used as a loading control. (B) RT-PCR analysis. Relative PD-L1 mRNA level was normalized to the expression of the GAPDH gene. (C,D) Immunocytochemistry of PD-L1 membrane expression and its quantification. Scale bar = 50 μm. Data are presented as mean±SD for WB and RT-PCR; mean±SEM for immunocytochemistry. n = 3. * p < 0.05, ** p < 0.01 compared to control. # p < 0.05, ## p < 0.01 compared to IFNγ and TNFα combined treatment.