Treatment of colon and pancreatic cancer cells with DIM-C-pPhBr or 2,2′-diMeDIM-C-pPhBr induces apoptosis and ER stress (37
) and results in show that after treatment of SW480 cells with these compounds for 24 hr, there was significant concentration-dependent decrease in cell proliferation. Both C-DIMs decreased cell numbers at concentrations of 10 or 15μM and at a concentration of 5μM, DIM-C-pPhBr also inhibited SW480 cell growth, whereas 2,2′-diMeDIM-C-pPhBr was inactive. show that both compounds also induce caspase-dependent PARP cleavage (15 and 20 μM) and at these same concentrations there was a parallel decrease in survivin expression in SW480 cells. In addition, we also observed that both C-DIM compounds decreased survivin mRNA expression after treatment for 24 hr ().
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 (more ...)
DIM-C-pPhBr and 2,2′-diMeDIM-C-pPhBr also inhibited Panc28 pancreatic cancer cell growth () and significant inhibition was observed at concentrations of 15–20 um. DIM-C-pPhBr and 2,2′-diMeDIM-C-pPhBr also induced PARP cleavage and decreased survivin protein levels in Panc28 cells () and this was also accompanied by decreased survivin mRNA levels. All of these responses were observed after treatment of SW480 or Panc28 cells for 24 hr. IC50 values for growth inhibition after treatment with DIM-C-pPhBr and 2,2′-diMeDIM-C-pPhBr were 10.8 and 8.3 μM (SW480) and 9.4 and 8.8 μM (Panc28), respectively.
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 (more ...)
We also investigated the time dependent effects of DIM-C-pPhBr and 2,2′-diMeDIM-C-pPhBr on survivin protein and cleaved PARP expression using a relatively high concentration (20 μM) of both compounds in order to determine differences between the temporal expression of both proteins. In SW480 cells, 20 μM DIM-C-pPhBr and 2,2′-diMeDIM-C-pPhBr decreased levels of survivin protein within 2 hr after treatment, whereas cleaved PARP protein was observed only after treatment for 12 hr (). Similar results were observed in Panc28 cells (); however, the extent of survivin degradation after treatment for 2 hr was lower in Panc28 compared to SW480 cells. These results show that there was a lag between the loss of survivin and induction of cleaved PARP in both cell lines.
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 (more ...)
illustrate the effects of 20 μM DIM-C-pPhBr and 2,2′-diMeDIM-C-pPhBr on survivin mRNA levels in SW480 and Panc28 cells. Both compounds either had no effect or induced survivin mRNA levels after treatment of SW480 or Panc28 cells for 2 hr; significant inhibition of survivin mRNA levels was observed in SW480 and Panc28 cells after 12 hr; however, the magnitude of survivin mRNA repression was more pronounced in the colon cancer cells. Thus, the effects of DIM-C-pPhBr and 2,2′-diMeDIM-C-pPhBr on survivin mRNA levels were observed at later time points compared to the rapid downregulation (within 2 hr) of survivin protein (). We also confirmed that DIM-C-pPhBr and 2,2′-diMeDIM-C-pPhBr decreased luciferase activity in SW480 cells transfected with pSurvivin-269, a construct containing the −269 to +49 region of the survivin promoter (). Results were not obtained in Panc28 cells due to low transfection efficiencies in this cell line. Since DIM-C-pPhBr and 2,2′-diMeDim-C-pPhBr decreased survivin protein but not mRNA levels within 2 hr after treatment, we investigated the effects of these compounds alone and in combination with the proteasome inhibitor MG132 after treatment of SW480 cells for 4 hr (). The results show that C-DIM-dependent downregulation of survivin protein was partially reversed by the proteasome inhibitor, suggesting that activation of proteasomes contributed to the early decrease in survivin expression.
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 (more ...)
Since DIM-C-pPhBr and 2,2′-diMeDIM-C-pPhBr inhibit survivin protein expression, the interactions of these compounds with radiotherapy were investigated. illustrates the effects of γ-radiation on proliferation of SW480 cells. Cells were administered doses of 2, 5 and 10 Gy using a Theatron 80 cobalt-60 teletherapy instrument and the effects of radiation on cell growth were determined after 24 and 48 hr. This cell line was responsive to radiation-induced inhibition of cell growth and after 24 hr, significant inhibition was observed using 5 and 10 Gy (but not 2 Gy) and all three doses of radiation inhibited cell growth after 48 hr. Using a similar protocol, γ-radiation also inhibited Panc28 cell proliferation (); however, this cell line was clearly more resistant to radiotherapy than SW480 cells over this time period and significant growth inhibition was observed only after 24 hr. The effects of γ-radiation on survivin expression were also investigated in SW480 () and Panc28 () cells and in both cell lines, 5 and 10 Gy induced survivin expression after radiation for 24 hr, whereas induction of survivin was either decreased or not observed after 48 hr. γ-Radiation also induced survivin and decreased proliferation of Panc1 and L3.6pl pancreatic cells and both cell lines were more responsive than Panc28 cells to the antiproliferative activity of γ-radiation ().
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 (more ...)
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 (more ...)
The combined effects of γ-radiation and C-DIM compounds on survivin expression are summarized in . Treatment of SW480 cells with 20μM DIM-C-pPhBr or 2,2′-diMeDIM-C-pPhBr for 24 hr showed that the C-DIMs alone decreased survivin, γ-radiation alone increased survivin and C-DIMs in combination with radiation decreased radiation-induced survivin expression. Similar effects were observed on cell numbers in SW480 or Panc28 cells (); however, the high concentrations (20 μM) of the C-DIM alone significantly decreased cell proliferation so that the interactions with γ-radiation were not apparent. We also examined the effects of lower concentrations of DIM-C-pPhBr and 2,2′-diMeDIM-C-pPhBr (5, 10 and 15 μM) on inhibition of γ-radiation-induced induction of survivin in SW480 cells (). Inhibition was only observed using 15 μM concentrations of C-DIMs and these concentrations alone also decreased levels of survivin protein. These results demonstrate that combinations of C-DIMs plus γ-radiation decrease radiation-induced survivin which plays a role in radioresistance.
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 (more ...)