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. 2008 Dec 15;14(24):8143-51.
doi: 10.1158/1078-0432.CCR-08-1539.

Nuclear factor-kappaB p65/relA silencing induces apoptosis and increases gemcitabine effectiveness in a subset of pancreatic cancer cells

Xue Pan  1 , Thiruvengadam Arumugam, Tameyoshi Yamamoto, Pavel A Levin, Vijaya Ramachandran, Baoan Ji, Gabriel Lopez-Berestein, Pablo E Vivas-Mejia, Anil K Sood, David J McConkey, Craig D Logsdon
Affiliations

Nuclear factor-kappaB p65/relA silencing induces apoptosis and increases gemcitabine effectiveness in a subset of pancreatic cancer cells

Xue Pan et al. Clin Cancer Res. .

Abstract

Purpose: Nuclear factor kappaB (NFkappaB) activity may increase survival and protect cancer cells from chemotherapy. Therefore, NFkappaB activity may be prognostic, and inhibition of NFkappaB may be useful for pancreatic cancer therapy. To test these hypotheses, we examined NFkappaB activity and the effects of inhibiting NFkappaB in several pancreatic cancer cell lines with differing sensitivities to gemcitabine.

Experimental design: The gemcitabine sensitivity of pancreatic cancer cell lines BxPC-3, L3.6pl, CFPAC-1, MPanc-96, PANC-1, and MIA PaCa-2 were determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and fluorescence-activated cell sorting assays. NFkappaB levels were determined by electrophoretic mobility shift assay and reporter assays. The effects of gemcitabine on NFkappaB activity were determined in vitro and in vivo. NFkappaB was inhibited by silencing of the p65/relA subunit using small interfering RNA in vitro and by neutral liposomal delivery of small interfering RNA in vivo, and the effects were evaluated on gemcitabine sensitivity.

Results: The cell lines L3.6pl, BxPC-3, and CFPAC-1 were sensitive, whereas MPanc-96, PANC-1, and MIA PaCa-2 were resistant to gemcitabine. No significant correlation was observed between basal NFkappaB activity and gemcitabine sensitivity. Gemcitabine treatment did not activate NFkappaB either in vitro or in vivo. Silencing of p65/relA induced apoptosis and increased gemcitabine killing of all gemcitabine-sensitive pancreatic cancer cells. No significant effects, however, were observed on gemcitabine-resistant pancreatic cancer cell lines either in vitro or in vivo.

Conclusions: NFkappaB activity did not correlate with sensitivity to gemcitabine. Silencing of p65/relA was effective alone and in combination with gemcitabine in gemcitabine-sensitive but not gemcitabine-resistant pancreatic cancer cells. Thus, NFkappaB may be a useful therapeutic target for a subset of pancreatic cancers.

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Conflict of interest statement

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Figures

Fig. 1
Fig. 1
Pancreatic cancer cells have differing levels of native resistance to gemcitabine. Human pancreatic cancer cell lines PANC-1, MPanc-96, MIA PaCa-2, CFPAC-1, BxPC-3, and L3.6pl were treated with increasing concentrations of gemcitabine (0–100 µmol/L) for 72 h. The viabilities indicated on the y axis were determined by MTS assays and normalized to control. Data shown are means ± SE for n = 3 independent experiments.
Fig. 2
Fig. 2
Basal levels of NFκB do not correlate with gemcitabine sensitivity. A, NFκB nuclear binding was analyzed in human pancreatic cancer cell lines. Nuclear extracts from six pancreatic cancer cell lines and human pancreatic duct epithelial cells were prepared and EMSA using a labeled oligonucleotide (Oligo) containing a consensus κ B binding site was done. TBP was used as a loading control for quality and quantity of cell extracts. Negative control, 32P-labeled NFκB Oligo without HeLa nuclear extract; positive control, HeLa nuclear extract and 32P-labeled NFκB Oligo; specific competitor, HeLa nuclear extract with 32P-labeled NFκB Oligo plus unlabeled NFκB Oligo; nonspecific competitor, HeLa nuclear extract with 32P-labeled NFκB Oligo plus unlabeled Oct1 Oligo. B, NFκB transcriptional activity was estimated using an NFκB reporter assay. Pancreatic cancer cells were coinfected with an NFκB reporter expressing firefly luciferase and a control lentivirus expressing renilla luciferase, and stable populations were developed. Basal NFκB transcriptional activity determined as a ratio of firefly and Renilla luciferase signals and presented as a comparison with the level measured in human pancreatic duct epithelial cells. As a positive control, all cell lines were also challenged with TNF-α (10 ng/mL) for 6 h and the level of NFκB activity was determined. Data shown are means ± SE for three independent experiments.
Fig. 3
Fig. 3
Gemcitabine treatment does not influence NFκB activity in pancreatic cancer cells in vitro or in vivo. A, two gemcitabine sensitive (L3.6pl and BxPC-3) and two gemcitabine resistant (MPan-96 and PANC-1) cell lines were treated with indicated concentrations of gemcitabine for 24 h in vitro and activity of an NFκB reporter was analyzed. B, the most gemcitabine-sensitive cell line (L3.6pl) and the most resistant (PANC-1) were implanted orthotopically in nude mice, allowed to form tumors, and then the level of NFκB reporter activity was measured before and 24, 48, 72 h after treatment with either a low dose (50 mg/kg) or a high dose (200 mg/kg) of gemcitabine. All measurements are shown as a percentage of basal and are means ± SE for three independent experiments.
Fig. 4
Fig. 4
siRNA silencing of p65/rel A was highly effective in vitro. A, to examine the effectiveness of p65/relA silencing on NFκB activity in the cancer cells, PANC-1 cells expressing NFκB luciferase gene were transfected with control siRNA and relA siRNA. After 72 h, cells were treated with or without TNF-α (10 ng/mL) and bioluminescent imaging was used to estimate NFκB reporter activity. B, Western blotting was conducted 72 h after treatment with control siRNA or siRNA against p65/relA in the six cell lines. Full-length gels are presented in Supplementary Fig. S1. Columns, mean for three experiments; bars, SE. *, P < 0.05, versus control.
Fig. 5
Fig. 5
Silencing of p65/relA is effective against gemcitabine-sensitive but not resistant cells. A, effects of gemcitabine on cell viability of pancreatic cancer cell lines after silencing of relA/p65. Human pancreatic cancer cell lines BxPC-3, L3.6pl, CFPAC-1, MPanc-96, PANC-1, and MIA PaCa-2 were treated with different concentrations of gemcitabine for 48 h. The cell numbers indicated on the y axis were determined by MTS assays and normalized to control as described in Materials and Methods. B, cell death assay for measuring apoptosis induced by siRNAp65, gemcitabine, and combination. Cells were plated at an equal density and the sub-G1 faction was assessed by fluorescence-activated cell sorting analysis after propidium iodide staining in BxPC-3 and Mpanc-96 cells. Data are means ± SE for n = 3–5 experiments.
Fig. 6
Fig. 6
Liposomal delivery of siRNA against p65/relA effectively inhibits NFκB activity and it also effectively inhibits growth of gemcitabine-sensitive pancreatic cancer cells but not gemcitabine-resistant pancreatic cancer cells in vivo. A, tumors were formed in nude mice with MPanc-96 cells stably expressing the NFκB reporter. The mice were treated with or without neutral liposomes bearing siRNAp65 and the effects on bioluminescence imaging indicated significant reduction. B, tumors were formed in nude mice with MPanc-96/BxPC-3 cells stably expressing a constitutively regulated luciferase for analysis of tumor formation. Bioluminescent imaging was done weekly to assess tumor growth. C, after 5 wk the weights of the pancreata including the primary tumors were quantified. Columns, mean for five animals; bars, SE. *, P < 0.05, versus control.
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