Requirement of nuclear factor κB for Smac mimetic-mediated sensitization of pancreatic carcinoma cells for gemcitabine-induced apoptosis

Abstract

Defects in apoptosis contribute to treatment resistance and poor outcome of pancreatic cancer, calling for novel therapeutic strategies. Here, we provide the first evidence that nuclear factor (NF) κB is required for Smac mimetic-mediated sensitization of pancreatic carcinoma cells for gemcitabine-induced apoptosis. The Smac mimetic BV6 cooperates with gemcitabine to reduce cell viability and to induce apoptosis. In addition, BV6 significantly enhances the cytotoxicity of several anticancer drugs against pancreatic carcinoma cells, including doxorubicin, cisplatin, and 5-fluorouracil. Molecular studies reveal that BV6 stimulates NF-κB activation, which is further increased in the presence of gemcitabine. Importantly, inhibition of NF-κB by overexpression of the dominant-negative IκBα superrepressor significantly decreases BV6- and gemcitabine-induced apoptosis, demonstrating that NF-κB exerts a proapoptotic function in this model of apoptosis. In support of this notion, inhibition of tumor necrosis factor α (TNFα) by the TNFα blocking antibody Enbrel reduces BV6- and gemcitabine-induced activation of caspase 8 and 3, loss of mitochondrial membrane potential, and apoptosis. By demonstrating that BV6 and gemcitabine trigger a NF-κB-dependent, TNFα-mediated loop to activate apoptosis signaling pathways and caspase-dependent apoptotic cell death, our findings have important implications for the development of Smac mimetic-based combination protocols in the treatment of pancreatic cancer.

Figure 1

BV6 enhanced gemcitabine-induced loss of viability. Panc1 (left) and MiaPaCa2 (right) cells were treated with indicated concentrations of gemcitabine (A), etoposide (B), doxorubicin (C), and cisplatin (D) and/or 2 µM BV6 for 72 hours. Cell viability was assessed by MTT assay and is calculated as percentage of untreated cells. Means ± SEM of three independent experiments performed in triplicate are shown. *P < .01 comparing samples in the presence or absence of BV6.

Figure 2

BV6 enhances gemcitabine-induced apoptosis. (A) DNA fragmentation. Panc1 (left) and MiaPaCa2 (right) cells were treated with indicated concentrations of gemcitabine and/or 2 µM BV6 for 72 hours. Apoptosis was determined by FACS analysis of DNA fragmentation of propidium iodide-stained nuclei. Means ± SEM of three independent experiments performed in triplicate are shown. *P < .01 comparing samples in the presence or absence of BV6. (B) Caspase cleavage. Panc1 (left) and MiaPaCa2 (right) cells were treated with 100 nM (Panc1) or 66 nM (MiaPaCa2) gemcitabine and/or 2 µM BV6 for indicated time points. Cleavage of caspase 8 and 3 was assessed by Western blot analysis. β-Actin served as loading control. One representative Western blot of two independent experiments is shown.

Figure 3

BV6- and gemcitabine-induced apoptosis is caspase- and TNFα-dependent. (A, B) Panc1 cells were treated for 72 hours with 100 nM gemcitabine and/or 2 µM BV6 and/or 20 µM zVAD.fmk, 30 µM necrostatin 1, 5 µg/ml Enbrel. Cell viability was assessed by MTT assay (A). Apoptosis was determined by FACS analysis of DNA fragmentation of propidium iodide-stained nuclei (B). Means ± SEM of three independent experiments performed in triplicate are shown. *P < .01. (C) Panc1 cells were treated for 48 hours with 100 nM gemcitabine and/or 2 µM BV6. TNFα protein levels in supernatants were determined by ELISA, and fold increase in TNFα secretion relative to untreated control is shown. Means ± SD of three experiments performed in triplicate are shown. *P < .05 comparing BV6 treatment to untreated control. (D) Panc1 cells were treated 100 nM gemcitabine and/or 2 µM BV6 in the presence or absence of 5 µg/ml Enbrel for 48 hours. Cleavage of caspase 8 and 3 was assessed by Western blot analysis. One representative of two experiments is shown. (E) Panc1 cells were treated with 100 nM gemcitabine and/or 2 µM BV6 in the presence or absence of 5 µg/ml Enbrel for 48 hours. Loss of mitochondrial membrane potential (MMP) was assessed by flow cytometry. Means ± SEM of three independent experiments performed in triplicate are shown. *P < .01.

Figure 4

NF-κB activation by BV6. (A) Panc1 cells were treated with 100 nM gemcitabine and/or 2 µM BV6 for 4 or 24 hours. Stimulation with 10 ng/ml TNFα for 1 hour served as positive control. NF-κB activation was assessed by the analysis of NF-κB DNA binding by EMSA. One representative of three experiments is shown. (B) Panc1 cells were transiently transfected with firefly and Renilla luciferase gene constructs, treated with 100 nM gemcitabine and/or 2 µM BV6 for 24 hours, and analyzed by dual luciferase assay for induction of NF-κB transcriptional activity. Fold increase in luciferase activity relative to untreated control is shown. Means ± SD of three experiments performed in triplicate are shown. *P < .05 comparing BV6 treatment to untreated control. (C) Panc1 cells were treated with 100 nM gemcitabine and/or 2 µM BV6 for 24 hours. Protein expression of Bcl-X_L was assessed by Western blot analysis. β-Actin served as loading control.

Figure 5

Requirement of NF-κB for BV6- and gemcitabine-induced cell death. Panc1 cells were stably transduced with a vector containing IκBα-SR or empty control vector. (A) Expression of IκBα-SR was determined by Western blot analysis using. (B) NF-κB activation in control and IκBα-SR-overexpressing cells was determined by EMSA after stimulation with 10 ng/ml TNFα for 1 hour. (C) IκBα-SR-overexpressing and control cells were treated with 2 µM BV6 and/or indicated concentrations of gemcitabine (nM) for 72 hours. Cell viability was determined by MTT assay, and this is calculated as the percentage of untreated cells. Means ± SEM of three independent experiments performed in triplicate are shown. *P < .01.