1,1-Bis(3'-indolyl)-1-(p-bromophenyl)methane and related compounds repress survivin and decrease gamma-radiation-induced survivin in colon and pancreatic cancer cells

Abstract

1,1-Bis(3'-indolyl)-1-(p-bromophenyl)methane (DIM-C-pPhBr) and the 2,2'-dimethyl analog (2,2'-diMeDIM-C-pPhBr) inhibit proliferation and induce apoptosis in SW480 colon and Panc28 pancreatic cancer cells. In this study, treatment with 10-20 microM concentrations of these compounds for 24 h induced cleaved PARP and decreased survivin protein and mRNA expression in both cell lines. However, results of time course studies show that DIM-C-pPhBr and 2,2'-diMeDIM-C-pPhBr decrease survivin protein within 2 h after treatment, whereas survivin mRNA levels were decreased only at later time-points indicating activation of transcription-independent and -dependent pathways for downregulation of survivin. In addition, we also observed that gamma-radiation inhibited pancreatic and colon cancer cell growth and this was associated with enhanced expression of survivin after 24 (SW480) or 24 and 48 h (Panc28) and correlated with previous studies on the role of survivin in radiation-resistance. However, in cells co-treated with gamma-radiation plus DIM-C-pPhBr or 2,2'-diMeDIM-C-pPhBr, induction of survivin by gamma-radiation was inhibited after co-treatment with both compounds, suggesting applications for these drugs in combination cancer chemotherapy with gamma-radiation.

Figure 1

Effects of C-DIMs on SW480 cell proliferation and expression of survivin and cleaved PARP (c-parp). Concentration-dependent effects on cell proliferation (A) and survivin and c-parp protein expression (B and C). Cells were treated with DMSO and different concentrations of DIM-C-pPhBr or 2,2′-diMeDIM-C-pPhBr for 24 hr. Cells were counted (A) or whole cell lysates were analyzed by western blots as described in the Materials and Methods. (D) Survivin mRNA levels. Cells were treated with DMSO or 10–20 μM of C-DIM compounds, and survivin mRNA levels were determined by real time PCR as described in the Materials and Methods. A significantly (p < 0.05) decreased response is indicated (*). β-Actin served as a loading control for all western blots in Figures 1–6.

Figure 2

Effects of C-DIMs on Panc28 cell proliferation and expression of survivin and c-parp. Concentration-dependent effects on cell proliferation (A) and survivin and c-parp protein expression (B and C). Cells were treated with DMSO and different concentrations of DIM-C-pPhBr or 2,2′-diMeDIM-C-pPhBr for 24 hr. Cells were counted (A) or whole cell lysates were analyzed by western blots as described in the Materials and Methods. (D) Survivin mRNA levels. Cells were treated with DMSO or 10–20 μM of C-DIM compounds, and survivin mRNA levels were determined by real time PCR as described in the Materials and Methods. A significantly (p < 0.05) decreased response is indicated (*).

Figure 3

Time course effects of DIM-C-pPhBr (20 μM) and 2,2′-diMeDIM-C-pPhBr (20 μM) on survivin and c-parp expression. SW480 cells were treated with DIM-C-pPhBr (A) or 2,2′-diMeDIM-C-pPhBr (B) and Panc28 cells were treated with DIM-C-pPhBr (C) or 2,2-diMeDIM-C-pPhBr (D) for 2, 12 or 24 hr. DMSO treatment served as a vehicle control. Whole cell lysates were analyzed by western blots as described in the Materials and Methods.

Figure 4

C-DIM-dependent effects on survivin expression. Changes in survivin mRNA expression in SW480 (A) and Panc28 (B) cells. Cells were treated with 20 μM DIM-C-pPhBr or 2,2′-diMeDIM-C-pPhBr for 24 hr, and survivin mRNA levels were determined by real time PCR as described in the Materials and Methods. (C) Effects on the survivin promoter. SW480 cells were transfected with pSurvivin(269) and treated with DMSO or the C-DIM compounds. Luciferase activity was determined as described in the Materials and Methods. (D) Effects of MG132. SW480 cells were treated with DMSO, C-DIM compounds alone (20 μM), 10 μM MG132, or a combination of C-DIMs plus MG132 for 4 hr. Whole cell lysates were analyzed by western blots as described in the Materials and Methods. Significantly (p < 0.05) decrease mRNA levels or luciferase activity (compared to DMSO) is indicated (*).

Figure 5

Effects of γ-radiation of cell proliferation and survivin expression. SW480 (A) and Panc28 (B) cell growth and expression of survivin in SW480 (C) and Panc28 (D) cells. Cells were treated with DMSO (control) and irradiated with 2, 5 or 10 Gy for 24 or 48 hr, and cell number and survivin protein expression (from whole cell lysates) were determined as described in the Materials and Methods.

Figure 6

Interactions of γ-radiation and C-DIMs. (A) Effects of C-DIM on γ-radiation-induced survivin protein expression. Cells were treated with DMSO, C-DIMs (20 μM), radiation (10 Gy), or C-DIMs plus radiation for 24 hr. Whole cell lysates were analyzed by western blots as described in the Materials and Methods. Effects of C-DIMs and radiation on proliferation of SW480 (B) and Panc28 cells (C). Cells were treated with DMSO, C-DIMs and radiation as described in (A), and cell numbers were determined as described in the Materials and Methods. (D) Concentration-dependent effects of C-DIMs on γ-radiation-induced survivin protein expression. SW480 cells were treated with DMSO; 5, 10 or 15 μM DIM-C-pPhBr or 2,2′-diMeDIM-C-pPhBr alone; or in combination with γ-radiation for 24 hr. Whole cell lysates were analyzed by western blots as described in the Materials and Methods.

Figure 7

Effects of γ-radiation on growth of Panc1 (A) and L3.6pl (B) pancreatic cancer cells and expression of survivin in Panc1 (C) and L3.6pl (D) cells. Experiments were performed as described in the Materials and Methods. β-Actin was used as a protein loading control.