Figure 1.
HDAC expression in pancreatic cancer cell lines and the HPDE cells.
Protein extracts from log phase AsPC-1, BxPC-3, PANC-1, HPAC, MIAPaCa-2, CFPAC-1, Capan-1, and the HPDE cells were subjected to Western blots probed by anti-HDAC or -β-actin antibody, as described in the Materials and Methods. The class I HDACs (1, 2, 3, and 8) were detected in all the cell lines though the levels were variable. In general, the levels of the class I HDACs in the HPDE cells were relatively lower compared to the majority of the pancreatic cancer cell lines. Interestingly, the majority of class IIa HDACs (except for HDAC5) were detected in almost all the pancreatic cancer cell lines but not in the HPDE cells. In contrast, HDACs 6 and 10 were detected in all the cell lines and their levels in the HPDE cells were comparable (if not higher) to those in the cancer cell lines.
Figure 2.
HDACI sensitivities in pancreatic cancer cell lines and the HPDE cells.
Panels A–C: PANC-1 cells were harvested and lysed after incubation with a range of concentrations of MGCD0103 (0–1.0 µM), MC1568 (0–10 µM), or Tubastatin A (0–4 µM) for 96 h. Soluble proteins were analyzed on Western blots probed by anti-acetylated (ac)-H4, -H4, -ac-tubulin, or –β-actin antibody. Panel D: AsPC-1, BxPC-3, PANC-1, or the HPDE cells were cultured at 37°C for 96 h in complete medium in 96-well plates, with a range of concentrations of MGCD0103, MC1568, or Tubastatin A, and cell viabilities were determined using the MTT reagent and a visible light microplate reader. The IC50 values were calculated as the concentrations of drug necessary to inhibit 50% growth compared to control cells cultured in the absence of drug. The data are presented as mean values ± standard errors from at least 3 independent experiments. MG, MGCD0103; MC, MC1568; TA, Tubastatin A. The same abbreviations were used throughout the study unless otherwise stated.
Figure 3.
Synergistic interactions in inducing growth arrest between MGCD0103 and MC1568 or Tubastatin A in BxPC-3 or PANC-1 cells.
Panels A and B: MGCD0103 IC50s of BxPC-3 or PANC-1 cells were determined in the absence or presence of MC1568 (panel A) or Tubastatin A (panel B) treated simultaneously. * indicates p<0.05, while ** indicates p<0.005. Panels C–F: Standard isobologram analysis of inhibition of PANC-1 (panels C&D) or BxPC-3 (panels E&F) cell growth by MGCD0103 and MC1568 (panels C&E) or Tubastatin A (panels D&F). The IC50 values of each drug are plotted on the axes; the solid line represents the additive effect, while the points represent the concentrations of each drug resulting in 50% inhibition of growth. Points falling below the line indicate synergism between drug combinations whereas those above the line indicate antagonism. Panels G and H: shRNA stable clones for a negative control (NTC), HDAC4, and HDAC6 were generated in PANC-1 cells. Expression levels of HDAC4, HDAC6, ac-H4 and ac-alpha-tubulin were determined by western blots (Panel G). Sensitivity to MGCD0103 of these shRNA stable clones was determined by MTT assays. ** indicates p<0.005.
Figure 4.
Interactions in inducing growth arrest between MGCD0103 and MC1568 or Tubastatin A in the normal HPDE cells.
Panels A and B: MGCD0103 IC50s of HPDE cells were determined in the absence or presence of MC1568 (panel A) or Tubastatin A (panel B) treated simultaneously. Panel C: Standard isobologram analysis of inhibition of the HPDE cell growth by MGCD0103 and Tubastatin A. The IC50 values of each drug are plotted on the axes; the solid line represents the additive effect, while the points represent the concentrations of each drug resulting in 50% inhibition of growth. Points falling below the line indicate synergism between drug combinations whereas those above the line indicate antagonism.
Figure 5.
Synergistic interactions in inducing cell death between MGCD0103 and MC1568 or Tubastatin A in BxPC-3 or PANC-1 cells.
Three hundred PANC-1 cells or five hundred BxPC-3 cells were plated in 100 mm dishes 1 day prior to the treatments with variable concentrations of MGCD0103, MC1568, or Tubastatin A, alone (panels A and B) or in combination (panels C and D) for 96 h. The drugs were then washed out, and the cells were cultured in drug-free complete medium for up to 3 weeks. Colonies were visualized by coomassie blue staining and counted. Results are presented as mean percentages ± standard errors relative to untreated control cells from three independent experiments. * indicates p<0.05, while ** indicates p<0.005.
Figure 6.
Panels A–D: Effects of class I- and class II-selective HDACIs on apoptosis and cell cycle progression in BxPC-3 and PANC-1 cells.
PANC-1 (panels A and B) or BxPC-3 (panels C and D) cells were treated with MGCD0103, MC1568, or Tubastatin A, alone or combined for 96 h. The cells were harvested and subjected to flow cytometry analysis to measure both baseline and HDACI-induced apoptosis (panels A and C) and cell cycle distribution (panels B and D). The experiments were repeated three times, and results are presented as means ± standard errors of triplicates from one representative experiment. ** indicates p<0.005. Panels E and F: Effects of class I- and class II-selective HDACIs on p21 expression and DNA DSBs in BxPC-3 and PANC-1 cells. PANC-1 (panel E) or BxPC-3 (panel F) cells were treated with MGCD0103, MC1568, or Tubastatin A, alone or combined for 96 h. The cells were harvested and soluble proteins extracted and subjected to Western blotting to measure p21 and γH2AX levels. β-actin was used as the loading control.