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Complementary effects of HDAC inhibitor 4-PB on gap junction communication and cellular export mechanisms support restoration of chemosensitivity of PDAC cells
O Ammerpohl, A Trauzold, B Schniewind, U Griep, C Pilarsky, R Grutzmann, H-D Saeger, O Janssen, B Sipos, G Kloppel and H Kalthoff
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Figure 1.
4-phenylbutyrate inhibits HDAC activity in pancreatic cancer cell lines. T3M-4 and BxPc3 cells were untreated or treated with 2.0 mM 4-PB for 48 h. Histone deacetylases activity of untreated cells was determined colorimetrically and set to 100%. The relative HDAC activity in treated cells was calculated. Results from five measurements are presented (mean
s.e.m). The observed inhibition proved to be highly significant (t-test; P<0.0001).
Figure 2.
4-phenylbutyrate reduced cell number of pancreatic carcinoma cell lines in vitro. Panc 1, T3M-4, COLO 357, BxPc3, primary human fibroblasts or H6c7 cells were cultivated in the absence (black square) or presence of increasing 4-PB concentrations (0.5 mM (triangle), 1.0 mM (inverted triangle), 2.0 mM (diamond), 5.0 mM (circle)) up to 10.0 mM (white square). After 0, 24, 48 and 72 h, the cell number per 1 ml medium was determined from every sample by cell counting using the CASY1 TT-cell counter. Mean and s.e.m from at least three experiments are presented. To document morphological changes, a photo from each cell line taken after 48 h of treatment with 5.0 mM 4-PB (lower photo) and a corresponding untreated control (upper photo) is presented, respectively.
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Figure 3.
4-phenylbutyrate induced apoptosis and cell cycle arrest of pancreatic cell lines in a concentration-dependent manner. (A) Panc 1, BxPc3, T3M-4 and COLO 357 cells were treated with different concentrations of 4-PB for 48 h. Cells were trypsinised and stained with propidium iodide. The percentages of apoptotic cells (black), cells in G1-phase (grey) or S- and G2-phase (white) were determined by flow cytometry. The mean from at least three samples is presented. (B) ZVAD inhibited 4-PB induced apoptosis. Panc 1 and COLO 357 cells were treated either with 5.0 mM 4-PB alone or a combination of 5.0 mM 4-PB and 23 
g ml-1 zVAD for 72 h. Cells were trypsinised and stained with propidium iodide. The percentage of apoptotic cells was determined by flow cytometry. Prism4 was used to perform t-test analysis. Effects of zVAD treatment proved to be highly significant (P<0.0001).
Figure 4.
4-phenylbutyrate reduced cellular export and increased intercellular communication. (A) Pancreatic carcinoma cells either untreated, treated with 2.0 mM 4-PB, 5.0 mM 4-PB, 10.0 mM 4-PB or 100.0
M verapamil for 72 h were stained with Calcein-AM. The dye export was visualised by flow cytometry. The cellular export of calcein-AM was decreased by applying 4-PB in a dose-dependent manner (increase in fluorescence intensity) (B) Calcein-AM stained and unstained T3M-4 cells were co-cultured in the absence or presence of 4-PB (1,0 mM or 5,0 mM 4-PB as indicated) for 5 h. Cell cytometry was performed to distinguish unstained from calcein-AM stained cells. In the presence of 4-PB, the number of intermediately stained cells increased (right panel), whereas the number of unstained cells was reduced, showing intercellular dye transfer. The number of cells involved in dye transfer was calculated (% of 'intermediately' stained cells).
Figure 5.
4-phenylbutyrate and gemcitabine induced apoptosis synergistically due to activation of Caspase 8 and BID in a JNK-pathway-dependent manner. (A) Cells were either not treated (fine dotted), treated with gemcitabine (1.0 g ml-1, dotted), 4-PB (1.0 mM, dashed) or gemcitabine and 4-PB simultaneously for 48 h and utilised for JAM assay. Mean and s.d of the number of viable cells (compared to control) from at least three experiments are shown. Differences between the single drug and the combinatorial treatment proved to be significant (t-test). (B) BxPc3 or T3M-4 cells either not treated or treated with gemcitabine (1.0 g ml-1), 4-PB (1.0 mM), JNKi (SP600125, inhibitor of c-Jun N-terminale kinase, 20
M) or different combination of these drugs for 48 h as indicated in the legend were stained by annexin V/propidium iodide and employed for cell cytometry. Number of viable cells is presented. The gemcitabine-induced cell death (as single drug or in combination with 4-PB) could be inhibited by inhibition of JNK. (C) Peripheral blood mononuclear cells were isolated from two human donors (no. 1 and no. 2) as described in Materials and Methods. Cells were not treated or treated with gemcitabine, 4-PB or a combination of both for 20 h. Cells were collected, stained by annexin V/propidium iodide and FACS analysis was conducted. The number of viable cells is presented. (D) After isolating proteins from BcPc3 or T3M-4 cells not treated (-) or treated (+) with 1.0 mM 4-PB and/or 1.0 g ml-1 gemcitabine for 48 h, 20 g protein were used to conduct Western blot analysis using specific antibodies against Caspase 8, Bid, PARP, phosphorylated JNK/SAPK, whole JNK/SAPK or 
-actin as a control (p18: cleaving product of Caspase 8 activation).
