Concurrent inhibition of NF-kappaB, cyclooxygenase-2, and epidermal growth factor receptor leads to greater anti-tumor activity in pancreatic cancer

Abstract

Inactivation of survival pathways such as NF-kappaB, cyclooxygenase (COX-2), or epidermal growth factor receptor (EGFR) signaling individually may not be sufficient for the treatment of advanced pancreatic cancer (PC) as suggested by recent clinical trials. 3,3'-Diindolylmethane (B-DIM) is an inhibitor of NF-kappaB and COX-2 and is a well-known chemopreventive agent. We hypothesized that the inhibition of NF-kappaB and COX-2 by B-DIM concurrently with the inhibition of EGFR by erlotinib will potentiate the anti-tumor effects of cytotoxic drug gemcitabine, which has been tested both in vitro and in vivo. Inhibition of viable cells in seven PC cell lines treated with B-DIM, erlotinib, or gemcitabine alone or their combinations was evaluated using 3-(4,5-dimetylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Significant inhibition in cell viability was observed in PC cells expressing high levels of COX-2, EGFR, and NF-kappaB proteins. The observed inhibition was associated with an increase in apoptosis as assessed by ELISA. A significant down-regulation in the expression of COX-2, NF-kappaB, and EGFR in BxPC-3, COLO-357, and HPAC cells was observed, suggesting that simultaneous targeting of EGFR, NF-kappaB, and COX-2 is more effective than targeting either signaling pathway separately. Our in vitro results were further supported by in vivo studies showing that B-DIM in combination with erlotinib and gemcitabine was significantly more effective than individual agents. Based on our preclinical in vitro and in vivo results, we conclude that this multi-targeted combination could be developed for the treatment of PC patients whose tumors express high levels of COX-2, EGFR, and NF-kappaB.

Fig. 1

Cell viability of human pancreatic cancer (PC) cell lines treated with B-DIM, erlotinib (Erl), gemcitabine (Gem), and the combination evaluated by the MTT assay. AsPC-1, BxPC-3, COLO-357, L3.6pl, HPAC, MIA PaCa-2, and PANC-1 cells were treated with B-DIM (25 μM), erlotinib (2 μM), gemcitabine (10 nM), and the combination. There was a significant reduction in cell growth in the BxPC-3, COLO-357, and HPAC cells treated with B-DIM in combination with either erlotinib or gemcitabine or triple combination compared to cells treated with single agents. A trend for potentiation of growth inhibition by B-DIM was observed in L3.6pl cells when combined with erlotinib or gemcitabine, but no such potentiation was observed in AsPC-1, MIA PaCa-2, and PANC-1 cells.

Fig. 2

The level of COX-2, EGFR, pEGFR, and NF-κB activation was compared between a panel of seven pancreatic cancer cell lines (A). Expression of protein was assayed by Western blot analysis (upper panel), NF-κB activation was evaluated by the EMSA (lower panel). Isobologram plots for combination treatments with B-DIM (12.5, 25, and 37.5 μM), erlotinib (1, 2, and 3 μM), and gemcitabine (10, 20, and 30 nM) in BxPC-3, COLO-357, and HPAC cell line was evaluated by the MTT assay. CI, combination index (B). Cell survival of BxPC-3, cells treated with B-DIM (25 μM), erlotinib (2 μM), gemcitabine (10 nM), and the combinations were evaluated by the clonogenic assay (C). Photo micrographic differences in colony formation in BxPC-3 cell line untreated and treated are shown. [Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com.]

Fig. 3

Induction of apoptosis in PC cell lines treated with B-DIM, erlotinib (Erl), gemcitabine (Gem), and the combination, which was evaluated by the ELISA assay. Cells were treated with 25 μM B-DIM, 2 μM erlotinib, 10 nM gemcitabine, or the combination. There was a significant potentiation in the induction of apoptosis in BxPC-3, COLO-357, and HPAC cells treated with B-DIM in combination with erlotinib or gemcitabine and triple combination as compared to cells treated with either agent alone.

Fig. 4

The expression of EGFR, EGFR-p-tyrosine, COX-2, and NF-κB by Western blot analysis. BxPC-3, COLO-357, and HPAC human pancreatic cell lines were treated with B-DIM (25 μM), erlotinib (2 μM), gemcitabine (10 nM), or the combination. Significant down-regulation of all proteins was observed in cells treated with the double and triple combinations as compared to cells treated with either agent alone.

Fig. 5

Anti-tumor activity in L3.6pl (A) and COLO-357 (B) cells derived tumors. Changes in tumor weight showing efficacy of B-DIM, erlotinib, and gemcitabine combination treatment in L3.6pl (C) and COLO-357 (D) cells derived tumors, respectively.

Fig. 6

NF-κB DNA binding activity in nuclear extracts of randomly selected tumor tissues by EMSA in L3.6pl derived tumors (A; upper panel); quantification of NF-κB DNA binding activity (A; lower panel; n = 7). NF-κB DNA binding activity in nuclear extracts of randomly selected tumor tissues by EMSA in COLO-357 derived tumors (B; upper panel), quantification of NF-κB DNA binding activity (B; lower panel), and the inhibition of protein expression of COX-2, EGFR, and pEGFR in COLO-357 derived tumors (C).