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. 2012 Apr 15;1(4):295-307.
doi: 10.1242/bio.2012539. Epub 2012 Feb 10.

Targeting developmental regulators of zebrafish exocrine pancreas as a therapeutic approach in human pancreatic cancer

Nelson S Yee  1 , Weiqiang Zhou, Stephen G Chun, I-Chau Liang, Rosemary K Yee
Affiliations

Targeting developmental regulators of zebrafish exocrine pancreas as a therapeutic approach in human pancreatic cancer

Nelson S Yee et al. Biol Open. .

Abstract

Histone deacetylases (HDACs) and RNA polymerase III (POLR3) play vital roles in fundamental cellular processes, and deregulation of these enzymes has been implicated in malignant transformation. Hdacs and Polr3 are required for exocrine pancreatic epithelial proliferation during morphogenesis in zebrafish. We aim to test the hypothesis that Hdacs and Polr3 cooperatively control exocrine pancreatic growth, and combined inhibition of HDACs and POLR3 produces enhanced growth suppression in pancreatic cancer. In zebrafish larvae, combination of a Hdac inhibitor (Trichostatin A) and an inhibitor of Polr3 (ML-60218) synergistically prohibited the expansion of exocrine pancreas. In human pancreatic adenocarcinoma cells, combination of the HDAC inhibitor suberoylanilide hydroxamic acid (SAHA) and ML-60218 produced augmented suppression of colony formation and proliferation, and induction of cell cycle arrest and apoptotic cell death. The enhanced cytotoxicity was associated with supra-additive upregulation of the pro-apoptotic regulator BAX and the cyclin-dependent kinase inhibitor p21(CDKN1A). tRNAs have been shown to have pro-proliferative and anti-apoptotic roles, and SAHA-stimulated expression of tRNAs was reversed by ML-60218. These findings demonstrate that chemically targeting developmental regulators of exocrine pancreas can be translated into an approach with potential impact on therapeutic response in pancreatic cancer, and suggest that counteracting the pro-malignant side effect of HDAC inhibitors can enhance their anti-tumor activity.

Keywords: Exocrine pancreas; Histone deacetylases; Pancreatic cancer; RNA polymerase III; Suberoylanilide hydroxamic acid; Targeted therapy; Trichostatin A; Zebrafish.

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Conflict of interest statement

Competing interests: The authors declare no competing financial interests.

Figures

Fig. 1.
Fig. 1.. TSA at 165 nM induces maximal acetylation of histone H3 and near-maximal acetylation of histone H4.
Immunoblot analysis of acetylated histones H3 and H4. WT zebrafish larvae at 48 h.p.f. were incubated with TSA at various concentrations, DMSO, or no treatment, for 24 hours. Lane 1 (8.25 nM TSA), 2 (16.5 nM TSA), 3 (41.25 nM TSA), 4 (82.5 nM TSA), 5 (165 nM TSA), 6 (330 nM TSA), 7 (825 nM TSA), 8 (0.5% DMSO), and 9 (no treatment). Total protein was extracted from each group of larvae at 72 h.p.f., and analyzed by immunoblotting using the indicated antibodies. The intensity and area of each protein band was quantified by densitometric analysis. Each value represents the ratio of acetylated histone H3 (AcH3) to total histone H3, and acetylated histone H4 (AcH4) to H4, relative to that of control (no treatment).
Fig. 2.
Fig. 2.. TSA impairs growth and disrupts morphogenesis of exocrine pancreas in zebrafish larvae with hyperacetylation of nucleosomal histones.
WT zebrafish larvae were incubated in the absence or presence of 165 nM TSA (added at 48 h.p.f.) for 24 hours and then analyzed. (A) Exocrine pancreas (arrows) was analyzed by in situ hybridization using trypsin anti-sense riboprobes. Pancreatic acinar morphology by immunohistochemistry using anti-cadherin (Cad) antibodies, followed by transverse histological sectioning. e.p. exocrine pancreas; i, intestine. (B) The larvae were pulse-labeled with BrdU and analyzed by immunohistochemistry using anti-BrdU antibodies, followed by transverse histological sectioning. The number (#) of DAPI+ nuclei, the number (#) of BrdU+ nuclei, and the proportion of cells in S phase (% BrdU+ nuclei) were determined. Result is presented as the mean + s.d., and * indicates statistical significance, # trend of statistical significance. (C) Total protein was extracted and analyzed for acetylated and total histones H3 and H4 by immunoblotting. Anti-total histones H3 and H4 antibodies and anti-actin antibodies were used as internal controls.
Fig. 3.
Fig. 3.. TSA and ML-60218 synergistically inhibit expansion of exocrine pancreas in zebrafish by impeding cell cycle progression, with enhanced induction of histone acetylation and cyclin-dependent kinase inhibitors.
Starting at 48 h.p.f., WT larvae were incubated in the presence of TSA, ML-60218, TSA + ML-60218, or control (DMSO or untreated) for 24 hours and then analyzed. (A) Dorsal view of larvae with the exocrine pancreas (arrows) analyzed by whole mount in situ hybridization using anti-sense trypsin riboprobes. (B) Exocrine pancreatic epithelial cells in the S phase (upper panel) and morphometric analysis of cell growth (lower panel). The larvae were pulse-labeled with BrdU, processed for immunohistochemistry with anti-BrdU or anti-cadherin antibodies, followed by histological analysis. Each column indicates the mean proportion of BrdU+ nuclei or cell growth (area in µm2 per cell) in the exocrine pancreatic epithelia. (C) Total protein was extracted from the larvae and analyzed by immunoblotting using anti-acetylated histones H3 and H4 antibodies, and anti-total histones H3 and H4 antibodies. (D) Total RNA was extracted from the larvae and quantified for p21cdkn1a and p27cdkn1b mRNA using real-time PCR. Each column represents the mean p21cdkn1a or p27cdkn1b mRNA level normalized to gapdh mRNA and expressed as percentage of control from three independent experiments, with each real-time PCR conducted in triplicate samples. Bars represent s.e.m.; *P<0.05 considered statistically significant; NS, not statistically significant.
Fig. 4.
Fig. 4.. SAHA and ML-60218 inhibit anchorage-independent colony formation and induce cellular morphology consistent with cellular senescence and cell death.
(A) PANC-1 and BxPC-3 were treated with 5 µM SAHA, 100 µM ML-60218, 5 µM SAHA + 100 µM ML-60218, or untreated (control), and grown in soft agar for 14 days. Each column represents the mean number of colonies in each treatment group from 3 independent experiments, with each treatment group in triplicate; bars represent s.e.m. Statistical analysis was performed using Student's t-test to compare between each treatment and control except where indicated. *, P<0.05; #, P<0.005; @, P<0.0001. NS, not statistically significant. (B) Bright field images of the cells treated as described in (A) for 24 hours were captured under a phase contrast microscope. Green arrows are pointing at nuclear membrane blebbing; yellow arrows, cytoplasmic projections. Scale bar, 100 µm.
Fig. 5.
Fig. 5.. Combination of SAHA and ML-60218 produces augmented suppression of cellular proliferation with impaired cell cycle progression, enhanced apoptotic cell death; SAHA either alone or in combination with ML-60218 induces nuclear localization of survivin.
PANC-1 and BxPC-3 were treated with 5 µM SAHA, 100 µM ML-60218, 5 µM SAHA + 100 µM ML-60218, or untreated (control) for 48 hours and analyzed as follows. (A) Cellular proliferation by MTS assay. Each column represents the mean absorbance of three independent experiments, with each treatment in triplicate. Statistical analysis was performed using Student's t-test to compare between each treatment and control except where indicated. Bars represent s.e.m.; *, P<0.005; #, P<0.005; @, P<0.0005. NS, not statistically significant. (B) Flow cytometric analysis of cell cycle. The data shown are representative of three independent experiments with similar results. Non-specific sub-G0/G1 events were gated out for accurate analysis of cell cycle distribution curves in the viable cells. (C) Flow cytometric analysis of apoptosis in PANC-1. The proportion of apoptotic cells is indicated within the black border. The data shown are representative of three experiments with similar results. PI, propidium iodide. (D) Confocal microscopic analysis of survivin expression in BxPC-3 following immunocytochemistry. Survivin (green); cytoplasmic actin (red).
Fig. 6.
Fig. 6.. Effect of SAHA and ML-60218 on histone acetylation, BAX, and p21CDKN1A expression.
PANC-1 and BxPC-3 cells were incubated for 48 hours with 5 µM SAHA, 100 µM ML-60218, 5 µM SAHA + 100 µM ML-60218, 0.5% DMSO, or untreated (control) and analyzed by immunoblotting. (A) Expression of acetylated histones H3 and H4 was assessed using anti-acetylated histones H3 and H4 antibodies. Anti-total histones H3 and H4 antibodies were used as control. (B) Expression of BAX and p21CDKN1A protein was assessed using anti-BAX and anti-p21CDKN1A antibodies. Anti-actin antibodies were used to indicate equal amount of protein loaded. The data shown are representative of three independent experiments with similar results.
Fig. 7.
Fig. 7.. ML-60218-mediated suppression of SAHA-induced up-regulation of tRNA as a potential mechanism that contributes to the enhanced anti-cancer actions in pancreatic adenocarcinoma.
(A) The tRNAMet and tRNASer levels in PANC-1 and BxPC-3 cells treated for 48 hours as indicated are represented as % of control (untreated or DMSO). Each value is the mean + s.e.m. of three independent experiments, triplicate samples for each PCR, each PCR performed twice, and expressed as % relative to controls. Statistical analysis was performed comparing each experimental group with control, except where indicated. *P<0.05, statistically significant. #P<0.1, trend of statistical significance. NS, not statistically significant. (B) A working model that summarizes the enhanced cytotoxic actions of the combination of ML-60218 and either TSA or SAHA. i, inhibitor; AcH3, acetylated histone H3; AcH4, acetylated histone H4.
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