Suberoylanilide hydroxamic acid increases anti-cancer effect of tumor necrosis factor-α through up-regulation of TNF receptor 1 in lung cancer cells

Abstract

Suberoylanilide hydroxamic acid (SAHA) as a histone deacetylase (HDAC) inhibitor has anti-cancer effect. Here, we evaluated the effect of SAHA on HDAC activity and cell growth in many normal lung and cancer cells. We observed that the HDAC activities of lung cancer cells were higher than that of normal lung cells. SAHA inhibited the growth of lung cancer cells regardless of the inhibitory effect on HDAC. This agent induced a G2/M phase arrest and apoptosis, which was accompanied by mitochondrial membrane potential (MMP: ΔΨm) loss in lung cancer cells. However, SAHA did not induce cell death in normal lung cells. All tested caspase inhibitors prevented apoptotic cell death in SAHA-treated A549 and Calu-6 lung cancer cells. Treatment with tumor necrosis factor-alpha (TNF-α) enhanced apoptosis in SAHA-treated lung cancer cells through caspase-8 and caspase-9 activations. Especially, SAHA increased the expression level of TNF-α receptor 1 (TNFR1), especially acetylation of the region of TNFR1 promoter -223/-29 in lung cancer cells. The down-regulation of TNFR1 suppressed apoptosis in TNF-α and SAHA-treated lung cancer cells. In conclusion, SAHA inhibited the growth of lung cancer cells via a G2/M phase arrest and caspase-dependent apoptosis. SAHA also enhanced apoptotic effect of TNF-α in human lung cancer cells through up-regulation of TNFR1. TNF-α may be a key to improve anti-cancer effect of HDAC inhibitors.

Keywords: apoptosis; histone deacetylase; lung cancer; suberoylanilide hydroxamic acid; tumor necrosis factor-α.

Figure 1. Effect of SAHA on HDAC activities in human normal lung and cancer cells

(A) and (B) Graphs show the basal HDAC activities of lung cancer patients (A), normal lung and cancer cells (B). (C) Graph shows the cytosol and nuclear HDAC activities in normal lung and cancer cells. ^#p < 0.05 compared with A (IA). ^$p < 0.05 compared with A (IIIA). ^&p < 0.05 compared with HPF cells. *p < 0.05 compared with SAHA-untreated control group.

Figure 2. Effects of SAHA on cell growth in normal lung and cancer cells

Exponentially growing cells were treated with indicated concentrations of SAHA for 24, 48 and 72 hours. Graphs show cell growth in HSAEC (A), HBEC (B), HPF (C), A549 (D), SK-LU-1 (E), Calu-6 (F), HCC-95 (G), HCC-1588 (H), NCI-H460 (I), NCI-H1299 (J), NCI-H69 (K) and HCC-33 (L). *p < 0.05 compared with SAHA-untreated control group.

Figure 3. Effects of SAHA on cell cycle and cell death in normal lung and cancer cells

Exponentially growing cells were treated with indicated concentrations of SAHA for 24 hours. (A) Graphs show the cell cycle distributions in HSAEC (#4), NCI-H460 and Calu-6 cells. (B) and (C) Graphs show the percent of sub-G1 (B) and annexin V-FITC positive cells (C). *p < 0.05 compared with SAHA-untreated control group.

Figure 4. Effects of SAHA on mitochondrial membrane potential, apoptosis-related protein levels and caspase-3 activities in normal lung and cancer cells

Exponentially growing cells were treated with indicated concentrations of SAHA for 24 hours. (A) Representative images of JC-1 red, JC-1 green, DAPI (blue) and merge in SAHA-treated Calu-6 cells. Red fluorescent images indicate high MMP (ΔΨm). Green fluorescent images shows low ΔΨm. (B) Graph shows the rhodamine 123 negative (ΔΨm loss) cells. (C) The protein levels of PARP-1, c-PARP-1, Bax, Bcl-2 and β-actin in HSAEC (#4), A549 and Calu-6 cells. (D) Graph shows the caspase-3 activities in A549 and Calu-6 cells. *p < 0.05 compared with SAHA-untreated control group.

Figure 5. Effects of caspase inhibitors and TNF-family cytokines on cell growth and cell death in SAHA-treated normal lung and cancer cells

Exponentially growing cells were treated with 5 μM SAHA and 15 μM each caspase inhibitor, 10 ng/ml TNF-α, 10 ng/ml TRAIL and 10 ng/ml FasL for 24 hours. (A) and (B) Graphs show the percent of annexin V-FITC positive cells (A) and cell growth (B). (C) and (D) Graphs show the percent of annexin V-FITC positive cells (C) and LDH release compared with that in the control cells (D). *p < 0.05 compared with SAHA-untreated control group. ^#p < 0.05 compared with cells treated with SAHA only.

Figure 6. Effects of SAHA on TNFR1 expression in HCC-95 and NCI-H460 cells

Exponentially growing cells were treated with 5 μM SAHA for 24 hours. (A) Each figure shows a representative for TNFR1 expression in HCC-95 and NCI-H460 cells. (B) The protein levels of TNFR1, TNFR2 and GAPDH in HCC-95 and NCI-H460 cells. (C) ChIP assay using H3, Ac-H3 (K9) and AC-H3 (K27) antibodies, and primers for the TNFR1 promoter regions. NCI-H460 cells were transfected with nontarget control (CTR) siRNA or TNFR1 siRNA. After one day, cells were treated with 5 μM SAHA and 10 ng/ml TNF-α for 24 hours. (D) Graph shows the percent of annexin V-FITC positive cells. The inside figure indicates the protein levels of TNFR1 and GAPDH in NCI-H460 cells. ^#p < 0.05 compared with cells treated with SAHA only. ^$p < 0.05 compared with cells treated with SAHA and TNF-α.

Figure 7. Schematic diagram of SAHA-induced cell death in lung cancer cells