Combination of 12-O-tetradecanoylphorbol-13-acetate with diethyldithiocarbamate markedly inhibits pancreatic cancer cell growth in 3D culture and in immunodeficient mice

Abstract

The aim of the present study was to determine the effects of 12-O-tetradecanoylphorbol-13-acetate (TPA) and diethyldithiocarbamate (DDTC) alone or in combination on human pancreatic cancer cells cultured in vitro and grown as xenograft tumors in nude mice. Pancreatic cancer cells were treated with either DDTC or TPA alone, or in combination and the number of viable cells was then determined by trypan blue ecxlusion assay and the number of apoptotic cells was determined by morphological assessment by staining the cells with propidium idiode and examining them under a fluorescence microscope. Treatment with DDTC or TPA alone inhibited the growth and promoted the apoptosis of pancreatic cancer cells in a concentration-dependent manner. These effects were more prominent following treatment with TPA in combination with DDTC than following treatment with either agent alone in PANC-1 cells in monolayer cultures and in 3 dimensional (3D) cultures. The potent effects of the combination treatment on PANC-1 cells were associated with the inhibition of nuclear factor-κB (NF-κB) activation and the decreased expression of Bcl-2 induced by DDTC, as shown by NF-κB-dependent reporter gene expression assay and western blot analysis. Furthermore, treatment of nude mice with DDTC + TPA strongly inhibited the growth of PANC-1 xenograft tumors. The results of the present study indicate that the administration of TPA and DDTC in combination may be an effective strategy for inhibiting the growth of pancreatic cancer.

Figure 1

Structures of 12-O-tetradecanoylphorbol-13-acetate (TPA) and diethyldithiocarbamate (DDTC).

Figure 2

Effects of 12-O-tetradecanoylphorbol-13-acetate (TPA) and diethyldithiocarbamate (DDTC) alone or in combination on human pancreatic cancer cells. (A-C) PANC-1, MIA PaCa-2 and Bx-PC-3 cells were seeded at a density of 0.2×10⁵ cells/ml in cell culture dishes and incubated for 24 h. The cells were then treated with TPA or DDTC alone or in combination for 72 h. The number of viable cells was determined by the trypan blue exclusion assay. ^*P<0.001, compared to treatment with either agent alone. (D-G) Morphology of PANC-1 cells treated with TPA or DDTC alone or in combination as described above. Representative micrographs of PANC-1 cells in the (D) control, (E) DDTC-treated, (F) TPA-treated and (G) TPA + DDTC-treated groups are shown. (H-K) Morphology of PANC-1 cells in 3D culture. PANC-1 cells were seeded at a density of 0.5×10⁵ cells/ml in Matrigel in a 12-well plate (1 ml/well) and incubated for 2 h to allow the gel to solidify. DMEM was added on top of the gel (1 ml/well), and the cells were incubated for 24 h. The cells were then treated with TPA and DDTC alone or in combination once every other day for 10 days. Representative micrographs of PANC-1 cell 3D cultures in the (H) control, (I) DDTC-treated, (J) TPA-treated and (K) TPA + DDTC-treated groups are shown.

Figure 3

Effects of 12-O-tetradecanoylphorbol-13-acetate (TPA) and/or dieth-yldithiocarbamate (DDTC) on nuclear factor-κB (NF-κB) activity. PANC-1 cells were seeded at a density of 0.2×10⁶ cells/ml in medium in 60 mm culture dishes (5 ml/dish) and incubated for 24 h. The cells were then transfected with a NF-κB-luciferase construct using Lipofectamine™ 2000 (LF2000; Invitrogen Life Technologies). The cells were treated with TPA alone or in combination with DDTC for 24 h. The luciferase activity was determined using a lucif-erase assay kits (E1500; Promega). Each value represents the mean ± SE from 3 separate experiments. ^*P<0.01 compared to treatment with TPA alone.

Figure 4

Effect of 12-O-tetradecanoylphorbol-13-acetate (TPA) and/or dieth-yldithiocarbamate (DDTC) on the level of Bcl-2 in PANC-1 cells. PANC-1 cells were seeded at a density of 1×10⁵ cells/ml in medium and incubated for 24 h. The cells were then treated with TPA or DDTC alone or in combination for 48 h. Bcl-2 expression was measured by western blot analysis using an anti-Bcl-2 antibody (05-729; Millipore Corp.).

Figure 5

Effects of intraperitoneal injections of 12-O-tetradecanoylphorbol-13-acetate (TPA) or diethyldithiocarbamate (DDTC) alone or in combination on (A) the growth of PANC-1 tumors and (B) the body weight of NCr nude mice. Male NCr nude mice were injected subcutaneously with PANC-1 cells (2.0×10⁶ cells/mouse). Mice with established tumors (0.6-1.0 cm wide and 0.6-1.0 cm long) received daily intraperitoneal injections of TPA or DDTC alone or in combination for 28 days. Tumor size (length × width) and body weight were measured and expressed as a percentage of the initial tumor size or a percentage of the initial body weight. Each value represents the mean ± SE.

Figure 6

Effect of treatment 12-O-tetradecanoylphorbol-13-acetate (TPA) or diethyldithiocarbamate (DDTC) alone or in combination on the expression of proliferating cell nuclear antigen (PCNA) in PANC-1 tumors. Immunohistochemistry with a PCNA antibody was performed on the paraffin-embedded sections of PANC-1 tumors collected from the mice following treatment as described in Fig. 5. Representative micrographs of PCNA immunostaining in tumors from the (A) control, (B) TPA-treated, (C) DDTC-treated and (D) TPA + DDTC-treated groups are shown. PCNA positive cells were determined using a microscope (Nikon Optiphot) and (E) expressed as a percentage of PCNA-positive cells. Each value represents the mean ± SE. ^*P<0.01, compared to treatment with either agent alone.