HDAC2 attenuates TRAIL-induced apoptosis of pancreatic cancer cells

Abstract

Background: Pancreatic ductal adenocarcinoma (PDAC) is one of the most malignant tumors with a dismal prognosis and no effective conservative therapeutic strategies. Although it is demonstrated that histone deacetylases (HDACs), especially the class I HDACs HDAC1, 2 and 3 are highly expressed in this disease, little is known about HDAC isoenzyme specific functions.

Results: Depletion of HDAC2, but not HDAC1, in the pancreatic cancer cell lines MiaPaCa2 and Panc1 resulted in a marked sensitization towards the tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). Correspondingly, the more class I selective HDAC inhibitor (HDACI) valproic acid (VPA) synergized with TRAIL to induce apoptosis of MiaPaCa2 and Panc1 cells. At the molecular level, an increased expression of the TRAIL receptor 1 (DR5), accelerated processing of caspase 8, pronounced cleavage of the BH3-only protein Bid, and increased effector caspase activation was observed in HDAC2-depleted and TRAIL-treated MiaPaCa2 cells.

Conclusions: Our data characterize a novel HDAC2 function in PDAC cells and point to a strategy to overcome TRAIL resistance of PDAC cells, a prerequisite to succeed with a TRAIL targeted therapy in clinical settings.

Figure 1

HDAC2 depletion sensitizes PDAC cells towardsTRAIL. A) Western blot analysis of HDAC2 and HDAC1 48 hours after the transfection of MiaPaCa2 (upper panel) and Panc1 cells (lower panel) with a control siRNA or a HDAC2-specific siRNA. β-actin controls equal protein loading. B) MiaPaCa2 (left graph) and Panc1 cells (right graph) were transfected with a control siRNA or a HDAC2-specific siRNA. 48 hours after the transfection the cells were treated with increasing doses of TRAIL as indicated for additional 24 hours or left as an untreated control. Viability was determined using MTT assays (Student's t-test: * p < 0.05 versus controls). C) MiaPaCa2 (left graph) and Panc1 cells (right graph) were transfected with a control siRNA or a HDAC2-specific siRNA. 48 hours after transfection the cells were treated with increasing doses of TRAIL (6.25, 12.5, 25, 50 and 100 ng/ml) for additional 24 hours or left as an untreated control. Apoptotic cells were quantified by fluorescence microscopy after Hoechst staining (Student's t-test: * p < 0.05 versus controls). D) MiaPaCa2 and Panc1 cells were transfected with a control siRNA or a HDAC2-specific siRNA. 48 hours after the transfection the cells were treated with 25 ng/ml TRAIL for additional 24 hours or left as an untreated control. Cleaved PARP western blotting was used as an indirect measurement of caspase activity. β-actin controls equal protein loading.

Figure 2

The HDACI VPA sensitizes PDAC cells towards TRAIL-induced apoptosis. A) MiaPaCa2 (left graph) and Panc1 cells (right graph) were pre-treated with VPA or were left as an untreated control. After 48 hours the cells were treated with TRAIL or the combination of TRAIL and VPA for additional 24 hours or were left as an untreated control. TRAIL was used at concentrations of 6.25, 12.5, 25, 50 and 100 ng/ml. Viability was measured by MTT assays (Student's t-test: * p < 0.05 versus controls). B) MiaPaCa2 (left graph) and Panc1 cells (right graph) were pre-treated with VPA or were left as an untreated control. After 48 hours the cells were treated with TRAIL or the combination of TRAIL and VPA for additional 24 hours or were left as an untreated control. TRAIL was used at concentrations of 6.25, 12.5, 25, 50 and 100 ng/ml. Apoptotic cells were quantified by fluorescence microscopy after Hoechst staining (Student's t-test: * p < 0.05 versus controls).

Figure 3

HDAC1 is not involved in the regulation of TRAIL-induced apoptosis in PDAC cells. A) Western blot analysis of HDAC1 and HDAC2 48 hours after the transfection of MiaPaCa2 (left panel) and Panc1 cells (right panel) with a control siRNA or a HDAC1-specific siRNA. β-actin controls equal protein loading. B) MiaPaCa2 (left graph) and Panc1 cells (right graph) were transfected with a control siRNA or a HDAC1-specific siRNA. 48 hours after the transfection the cells were treated with 50 ng/ml TRAIL for additional 24 hours or left as an untreated control. Viability was determined using MTT assays (Student's t-test: * p < 0.05 versus controls).

Figure 4

HDAC2-dependent regulation of NOXA does not contribute to TRAIL sensitization. A) Quantitative NOXA and HDAC2 mRNA expression analysis in MiaPaCa2 cells after the transfection of a control, a HDAC2-specific or the combination of HDAC2- and NOXA-specific siRNAs. Total RNA was prepared 48 hours post-transfection. NOXA and HDAC2 mRNA levels were quantified using real-time PCR analysis and normalized to cyclophilin expression levels (Student's t-test: * p < 0.05 versus controls). B) MiaPaCa2 cells were transfected with a control siRNA, a HDAC2-specific siRNA or co-transfected with a HDAC2- and NOXA-specific siRNA as indicated. 48 hours after the transfection the cells were treated with 30 ng/ml TRAIL for additional 24 hours or left as an untreated control. Apoptotic cells were quantified by fluorescence microscopy after Hoechst staining (Student's t-test: * p < 0.05 versus controls).

Figure 5

HDAC2-dependent regulation of DR5 in MiaPaCa2 cells. MiaPaCa2 cells were transfected with a control or a HDAC2-specific siRNA as indicated. A) 48 hours after the transfection the cells were treated with 15 ng/ml TRAIL or a combination of TRAIL and actinomycin D (1 μg/ml) for additional 2 hours. Afterwards caspase 3/7 activity was measured (Student's t-test: * p < 0.05 versus controls). B) 48 hours after the transfection the cells were treated with 30 ng/ml TRAIL as indicated. Western blot determines caspase 8 (55/50 kD), caspase 8 cleavage products (migrating at 40/36 kD and 23 kD), cleaved PARP and HDAC2 expression. β-actin controls equal protein loading. C) MiaPaCa2 cells were treated as in B). The caspase 8 cleavage product migrating at 40/36 kD was quantified in control and HDAC2 siRNA transfected MiaPaCa2 cells 3 hours after the TRAIL treatment in four independent experiments (Student's t-test: * p < 0.05 versus controls). D) 48 hours after the transfection the cells were treated with 30 ng/ml TRAIL for 6 hours. Western blot determines DR5, DR4, Bid, c-Flip, XIAP, cIAP1, cIAP2, mcl1, bcl_XL, survivin and HDAC2 expression. β-actin controls equal protein loading. E) Panc1 (left graph) and MiaPaCa2 cells (right graph) were transfected as indicated. Western blot determines DR5 and HDAC2 expression 48 hours after the transfection. β-actin controls equal protein loading. F) Panc1 (left graph) and MiaPaCa2 cells (right graph) were transfected as indicated. 48 hours after the transfection DR5 surface expression was determined by FACS analysis (Student's t-test: * p < 0.05 versus controls).